Chemistry and Biology of Pteridines: 5 University of Konstanz, West Germany, April 14–18, 1975 [Reprint 2019 ed.] 9783110838053, 9783110059281

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Chemistry and Biology of Pteridines

Chemistry and Biology of Pten'dines Proceedings of the 5th International Symposium held at the University of Konstanz, West Germany, April 14-18,1975

Editor Wolfgang Pfleiderer

W G DE

Walter de Gruyter • Berlin • New York 1975

Editor Wolfgang Pfleiderer, Dr. rer. nat., Professor, Department of Chemistry, University of Konstanz, Konstanz, West-Germany

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of Congress

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International Symposium on Chemistry and Biology of Pteridines, 5th, University of Konstanz, 1975. Chemistry and biology of pteridines. Bibliography: p. X V I , 950 Includes index. I . Pteridines-Congresses. I. Pfleiderer, Wolfgang, 1927 II. Title. [ D N L M : 1. Pteridines-Congresses. W3 IN916u 1975c/ QD401 161 1975 c ] QP801. P69I57 1975 5 7 4 . V 9 2 4 75-31877 ISBN 3-11-005928-2

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Chemistry and biology of pteridines: proceedings of the 5. Internat. Symposium at the Univ. of Konstanz, West Germany, April 14—18, 1975/ed. by Wolfgang Pfleiderer. ISBN 3-11-005928-2 NE: Pfleiderer, Wolfgang [ H r s g . ] : Universität < K o n s t a n z >

© Copyright 1975 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form — by photoprint, microfilm, or any other means — nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Druckerei Gerike, Berlin. Binding: Liideritz & Bauer, Berlin. Printed in Germany.

Preface

The present volume contains the proceedings of the 5th International Symposium on "Chemistry and Biology of Pteridines" held at the University of Konstanz, West Germany, from April 14th - 18th, 1975. Pteridines, a widely distributed class of naturally occurring compounds, owe their exceptional position in the field of heterocyclic chemistry partly to their discovery as wing pigments of butterflies and mainly to their unusual chemical properties, their conspicuous fluorescence and their importance in metabolism. The history of pteridines starts in 1889 when Frederick Gowland Hopkins focussed attention for the first time on an interesting group of natural pigments present in butterfly wings. In a 15 years period of brilliant and laborious investigations by Heinrich Wieland, Clemens Schopf and co-workers (1925-1940) the first climax in pteridine chemistry was reached when Robert Purrmann (1940-1941) elucidated the chemical structures of three of the most common butterfly pigments, leucopterin, xanthopterin and isoxanthopterin. The recognition of a new naturally occurring heterocyclic ring system stimulated research in the pteridine field tremendously and led to another exciting discovery a few years later. In 1946 the structure of folic acid, a water-soluble growth factor in bacteria and an anti-pernicious anemia factor in animals could be determined and it was shown that this vitamin is also a pterin derivative with a p-aminobenzoylglutamic acid side-chain in the 6-position. The vast growing development of the pteridine field increased our knowledge of various aspects from the chemical, biochemical and biological point of view very much in the past 25 years and gave rise to the necessity for exchange of information on an international scientific level. Four International Pteridine Symposia have already been held - in 1952 in Paris, 1954 in London, 1962 in Stuttgart and 1969 in Toba/Japan - where syntheses, reactions, physical and chemical properties, biological roles in metabolism, interconversions of physiologically active components as well as isolation and structural elucidations of natural pteridine derivatives were discussed

VI in great detail. These meetings proved to be very successful in bringing together scientists from different fields and backgrounds but with the same interest, namely pteridines. There seems to be no other group of scientists devoted so much to their own field, to inter-disciplinary co-operation and exchange of experiences like the pteridinologists. It was therefore an enthusiastic response to the invitation to the Fifth International Pteridine Symposium at Konstanz after a six year intermission. The largest group so far of scientists interested in pteridines met at the Bodensee in April this year to discuss all aspects of their recent work and the outlook for future investigations in this field. I hope that the reading of these proceedings will stimulate and inspire not only the experts but also the newcomers who are cordially invited to take part in the fascinating chemistry and biology of pteridines. Thanks are due to Prof. M.Viscontini and Prof. A.Albert for their advice in organising this symposium and to all contributors

for their excellent

contributions, their manuscripts and their interesting discussions. My appreciation is also extended to my co-workers and my secretary Mrs. H.Bauer for their assistance in all matters concerning the symposium. The main credit and all our thanks must, however, be given to the "Stiftung Volkswagenwerk", whose generous financial support made this meeting possible. Further financial assistance from the "Gesellschaft der Freunde und Förderer der Universität Konstanz", the "Ciba-Geigy AG", Basel, and "Burroughs Wellcome Company", USA, is gratefully acknowledged. Finally acknowledgement is made to the staff of the "Verlag Walter de Gruyter", Berlin, for its valuable co-operation in preparing these proceedings . Konstanz, July 1975

Wolfgang Pfleiderer

Contents

Significant Steps in the Discovery and Application of P t e r i d i n e s . By A. Albert

1

Specific Inhibition of Dihydrofolate Biosynthesis - A new Approach to Chemotherapy. By H . C . S . Wood

27

Mammalian Dihydrofolate Reductase: P o r c i n e Liver Enzyme. By M. Poe, C. D. Bennett, D. Donoghue, J . M . Hirshfield, M . N . Williams and K. Hoogsteen

51

Kinetic Studies of E s c h e r i c h i a Coli Dihydropteroate Synthetase. By R. F e r o n e and S . R . Webb

61

Dihydropteroate Synthase: Purification by Affinity Chromatography and Mechanism of Action. By C . J . Suckling, J . R . Sweeney and H . C . S . Wood

73

The P r o p e r t i e s of Gamma Glutamyl Hydrolase (Conjugase) f r o m Bovine Liver. By P . B. Rowe, M. Silink and R. Reddel

85

The Purification of Riboflavin Synthetase by Affinity Chromatography using 7-Oxolumazines. By R. WriggLesworth, C.D. Ginger, R . J . Kulick and H. C. S. Wood

93

Interaction of Riboflavin Synthetase with Analogues of 6, 7-Dimethyl8-Ribityllumazine. B y G . W . E . P l a u t a n d R . L . Beach

101

Structure-Activity Relationships among P t e r i d i n e Derivatives and Related Quinazolines a s Inhibitors of Dihydrofolate Reductases. By J . J . McCormack

125

Spectral Studies of Thymidylate Synthetase. By R . L . Kisliuk, Sharma, R. P . L e a r y and N. Beaudette

133

R.K.

F o u r Folate Metabolizing Enzymes of Mouse Embryo F i b r o b l a s t s and L - C e l l s as tested during the Culture Cycle. By B. GrzelakowskaSztabert, W. Chmurzyhska and M. Landman

143

The Activity of the Cobalamine-dependent Methionine-Synthetase (5-Methyl-5, 6, 7, 8-Tetrahydrofolic Acid: Homocysteine M e t h y l - t r a n s f e r a s e ) in rapidly growing Human Cells and the Effect of some Folic Acid Derivatives and Analogues. By H. Sauer and W. Wilmanns

153

Vili The Role of Folate Binding Proteins in Folate Metabolism. By S. Waxman

165

Transport of Folate Compounds into Mammalian and Bacterial Cells. By F . M . Huennekens and G. B. Henderson

179

Transport of Folate Compounds through the Membrane of Humai Lymphoblastoid Cells. By D. Niethammer and R . C . Jackson

197

The Characterization of High Molecular Weight Complexes of Folic Acid in Mammalian Tissues. By M. Zamierowski and C. Wagner . .

209

The Enzymic Synthesis of P t e r i n s in Escherichia Coli. By G. M. Brown, J . Yim, Y. Suzuki, M. C. Heine, and F . F o o r

219

Biosynthesis of P t e r i n s in Mammalian Systems. By K. Fukushima, I. Eto, T. Mayumi, W. Richter, S. Goodson and T. Shiota

247

The Synthesis of P t e r i n s Catalyzed by Enzymes f r o m Drosophila melanogaster. By G. M. Brown and C . L . Fan

265

Biosynthesis of Biopterin in the Intact Rat and in Mouse Neuroblastoma Cells. By K. Buff and W. Dairman

273

Studies on the Biosynthesis of Riboflavin. By A. Bacher, R. Baur, U. Eggers, H. H ä r d e r s and H. Schnepple

285

B. Mailänder,

Studies on the Mechanism of Phenylalanine Hydroxylase: Detection of an Intermediate. By S. Kaufman

291

Effect of Pteridine Cofactor Structure on the Regulation of Phenylalanine Hydroxylase Activity. By J . E . Ayling and G. D. Helfand

305

Interrelationships between C - l - M e t a b o l i s m and Photosynthesis in Euglena Gracilis. By E . A . Cossins and K. L. Lor

321

Folate Derivatives and P a t t e r n s of C - l Metabolism in Neurospora Crassa, Wild Type, S e r - 1 and F o r m a t e Mutants. By G. Combepine, E . A . Cossins and P . Y . Chan

331

The Biosynthesis of Folic Acid and Pteridine Cofactor(s) and its Regulation. By K. Iwai and M. Kobashi

341

Reduced P t e r i n s as Possible Mediators in Cellular Electron T r a n s f e r . By H. Rembold

359

The Handling and Metabolism of Folates in the Rat and Man, with Special Relationship to Disease. By J . A. Blair

373

Red Blood Cell Polyglutamyl Folates in Vitamin B ^ Deficiency. By J . P e r r y , I. Chanarin and M. Lumb

407

Novel Urinary Metabolites of Folic Acid in the Rat. By P . A . Barford, and J . A. Blair

413

IX " P r u n e " / " K i l I e r - o f - P r u n e " : A Complementary Lethal System in Drosophila Melanogaster Affecting Pteridine Metabolism. By J . H . P . Hackstein

429

Kynurenine as Trigger in PterorhodLnsynthesis in Ephestia Kühniella Z. By K. Müller

437

Growth Stimulation by 6-Phenacylisoxanthopterin and Related Compounds. By Y. Iwanama

445

The Possible Role in Gene Regulation of an Isoxanthopterinbinding Protein f r o m Qncopeltus Embryos. By H. S. F o r r e s t and J . H. Smith . .

453

Poly--J-Glutamyl Chain Lengths in some Natural Folates and Contributions of Folic Acid synthesized by Intestinal Microflora to Rat Nutrition. By Ch.M. Baugh, E. Braverman and M.G. Nair

465

Tissue-Specific Synthesis of Methotrexate Polyglutamates in the Rat. By V.M. Whitehead, M.M. P e r r a u l t and S. Stelcner

475

A Convenient Synthesis of Methotrexate and Related Compounds. By J . A . Montgomery, J . D . Rose, C. Temple, J r . , and J . R . P i p e r . .

485

Methods for the Synthesis of Folic Acid Analogs, Substituted Folic Acid Analogs, Folic Acid and Folic Acid Conjugates. By P . K . Sengupta, E. Khalifa, J . H. Bieri and M. Viscontini 9 10 New Folate Analogs: Alterations in the C -N Region. By M.G. Nair, and Ch. M. Baugh

495 503

The Synthesis and Study of 10-Thia-10-Deaza-Analogs of Pteroic Acid, Folic Acid, and of Related Compounds: The Problem of Racemization in the Cyclization of Substituted 2-Amino-3-Cyanopyrazines. By H.G. Mautner, Y.H. Kim, Y. Gaumont and R. L. Kisliuk

515

Synthesis and P r o p e r t i e s of 5-Methyldihydrofolate By T. L. Deits, A. Rüssel, K. Fujii and J . M . Whiteley

525

A Coloured Oxidation Product f r o m Tetrahydrofolate. By B. Rimkus, L. J aenicke and H. Rüdiger

535

Pteridine Synthesis. By E . C . Taylor

543

The 7-Azapteridines. By D . J . Brown and R.K. Lynn

575

Enzymatic Oxidation of Pteridin-4-Ones and Related Compounds. By F . Bergmann, L. Levene and I. T a m i r

603

Synthesis and Structure of Dihydro- and Tetrahydroderivatives of P t e r i n 6, 7-Dicarboxylic Acid. By R. Mengel and W. P f l e i d e r e r

617

Direct Conversion of 4-Hydroxypteridines to their 4-Amino Analogs. By G . R . Gapski and J . M . Whiteley

627

High P r e s s u r e Liquid Chromatography of Substituted Pteridines and Tetrahydropteridines. By S.W. Bailey and J . E . Ayling

633

X

Electrochemistry of Pteridines. By H. Lund

645

Pteridone Mediators in the Electrolysis of Biological Macromolecules. By S. Kwee

671

Electrochemistry of 6. 7-Dioxo-Pteridines. By R. Gottlieb and W. Pfleiderer

681

Protonation and Covalent Hydration of Nitrogen Heterocycles A Carbon-13 NMR Study (1). By U. Ewers, A. Gronenborn, H. Günther and L. Jaenicke

687

Reindarstellung und NMR-spektroskopische Untersuchung von 5, 6, 7, 8, -TetrahydrofÖlsäure. Von W. Frick

695

Sterische Wechselwirkungen in substituierten Tetrahydropterinen. Von R. Weber

705

Kinetisch-Mechanistische Untersuchungen an 5-Formyl-6, 7-dimethyl5, 6, 7, 8-tetrahydropterin, 10-Formyl-folsäure, 5, 10-Methenyl-5, 6, 7, 8tetrahydrofolsäure und 5-Methyl-10-formyl- 5, 6, 7, 8 -tetrahydrofolsäure. Von J.H. Bieri

711

CNDO-Rechnungen an Pterin, 6, 7 -Dimethyl-7, 8-Dihydropterin und 5-Formyl-6, 7-Dimethyl-5, 6, 7, 8-Tetrahydropterin (1). Von J.H. B i e r i . .

721

H-D Exchange in Pteridin-4-ones. By F. Bergmann, I. Tamir, L. Levene and M. Rahat

725

Oxidation-Reduction Properties of Pterins. By K.G. Scrimgeour

731

Autoxidative Conversions of Tetrahydropteridines and seme Related Ringsystems. By H. I.X. Mager

753

Autoxidation of Tetrahydropterins. By A.J. Pearson and J.A. Blair . . . .

775

Synthesis of Thiolumazine Nucleosides. By I. Southon and W. Pfleiderer

783

Pteridine Nucleotides, Synthesis and Enzymic Studies. By H. Rokos, and G. Harzer

795

Biology of Pigmentation in Pieridae Butterflies. By H. Descimon

805

The Pterins of the Pieridae and their Biosynthesis: Metabolism of D-Erythro-Neopterin and its 7, 8-Dihydro Derivative in Colias Croceus. By H. Descimon ;

841

Occurence and Distribution of known Pterins in some Species of Diptera. By E. Cerioni, C. Contini and U. Laudani

851

Pteridines in the Mammalian Retina and Light Effects. By G. Cremer-Bartels

861

Les Pterines des Planctons Marins: Identification de Plusieurs Derives chez les Algues Unicellulaires. Sur la Distribution des Pterines chez les Crustaces Copepodes. Par A. Momzikoff

871

XI Russupteridine. Von C. H. Eugster und P . X . Iten

881

Aurodrosopterins in Eye Colour Mutants of Drosophila Melanogaster. By I. Schwinck

919

New Results about Drosopterins. By K. Rokos and W. PfLeiderer

931

Synthesis and Absolute Configuration of Sepiapterin. By W. Pfleiderer

941

List of Contributors

A. Albert, Australian National University, Research School of Chemistry, Canberra, Australia K.J.M. Andrews, Roche Products Ltd., Welwyn Garden City, Great Britain J. Ayling, University of California, Dept. of Biological Chemistry, School of Medicine, Los Angeles, U.S.A. A. Bacher, Institut für Mikrobiologie, Universität Hohenheim, StuttgartHohenheim, West Germany P.A. Barford, The University of Aston, Dept. of Chemistry, Birmingham, Great Britain Ch.M. Baugh, University of South Alabama, Dept. of Biochemistry, Mobile, Alabama, U.S.A. S.W. Bailey, University of California, School of Medicine, Dept. of Biological Chemistry, Los Angeles, California, U.S.A. F. Bergmann, The Hebrew University, Hadassah Medical School, Dept. of Pharmacology, Jerusalem, Israel B. Bezbaruah, Fachbereich Chemie, Universität Konstanz, Konstanz, West Germany J. Bieri, Institut für Organische Chemie, Universität Zürich, Zürich, Switzerland J.A. Blair, The University of Aston, Dept. of Chemistry, Birmingham, Great Britain P.H. Boyle, Trinity College, Dept. of Chemistry, Dublin, Ireland D.J. Brown, Australian National University, John-Curtin-School of Medical Research, Canberra City, Australia G.M. Brown, Massachusetts Institute of Technology, Dept. of Biology, Cambridge, Mass., U.S.A. K. Buff, Roche Institute of Molecular Biology, Nutley, N.J., U.S.A. G. Combepine, University of Geneve, Dêpt. de Biologie Végétale, Geneve, Switzerland B. Cooper, McGill University, Royal Victoria Hospital, Montreal, Canada E.A. Cossins, University of Alberta, Dept. of Botany, Edmonton, Alberta, Canada G. Cremer-Bartels, Universitäts-Augenklinik, Universität Münster, Münster, West Germany J.I. Degraw, Stanford Research Institute, Menlo Park, California, U,S.AH. Descimon, Ecole Normale Supérieure, Laboratoire de Zoologie, Paris, France

xrv E. Elstner, Universität Bochum, Fachbereich Biologie, Bochum, West Germany C.H. Eugster, Institut für Organische Chemie, Universität Zürich, Zürich, Switzerland U. Ewers, Institut für Organische Chemie der Universität Köln, Köln, West Germany R. Ferone, The Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, N.C., U.S.A. W. Frick, Institut für Organische Chemie, Universität Zürich, Zürich, Switzerland R. Gottlieb, Fachbereich Chemie, Universität Konstanz, Konstanz, West Germany B. Grzelakowska-Sztabert, Nencki Institute of Experimental Biology, Dept. of Cellular Biochemistry, Warsaw, Poland H. Günther, Institut für Organische Chemie der Universität Köln, Köln, West Germany J.H.P. Hackstein, Zoologisches Institut der Universität Köln, Köln, West Germany P. Hemmerich, Fachbereich Biologie, Universität Konstanz, Konstanz, West Germany G. Hennings, Max-Planck-Institut für Biochemie, Martinsried bei Minchen, West Germany F. Huennekens, Scripps Clinic and Research Foundation, Dept. of Biochemistry, La Jolla, California, U.S.A. P.X. Iten, Institut für Organische Chemie, Universität Zürich, Zürich, Switzerland K. Iwai, Research Institute for Food Science, Kyoto University, Uji, Kyoto, Japan Y. Iwanami, Sasaki Institute, Dept. of Chemistry, Tokyo, Japan T. Jones, Chester Beatty Research Institute, London, Great Britain S. Kaufman, Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland, U.S.A. R. Kisliuk, Tufts University, School of Medicine, Boston, Mass., U.S.A. K. Kobayashi, Ciba-Geigy (Japan) Ltd., Ohto Yono Saitama, Japan S. Kwee, Institute of Medical Biochemistry, University of Aarhus, Aarhus, Denmark T. Leigh, ICI Imperical Chemical Industries Limited, Chemistry Department, Macclesfield, Great Britain H. Lund, University of Aarhus, Dept. of Organic Chemistry, Aarhus, Denmark H.I.X. Mager, Delft University of Technology, Biochem. and Biophys. Laboratory, Delft, Holland H.G. Mautner, Tufts University, School of Medicine, Dept. of Biochemistry & Pharmacology, Boston, Mass. U.S.A.

XV

J. McCormack, University of Vermont, Pharmacology Dept., Burlington, Vermont, U.S.A. R. Mengel, Fachbereich Chemie, Universität Konstanz, Konstanz, West Germany A. Momzikoff, Institut Océanographique de Paris, Paris, France J. Montgomery, Southern Research Institute, Birmingham, Alabama, U.S.A. K. Müller, Zoologisches Institut der Universität Köln, Köln, West Germany M.G. Nair, University of South Alabama, College of Medicine, Dept. of Biochemistry, Mobile, Alabama, U.S.A. D. Niethammer, Universität Ulm, Universitäts-Kinderklinik, Ulm, West Germany J. Nixon, Wellcome Research Laboratories, Dept. of Medicinal Biochemistry, Research Triangle Park, N.C., U.S.A. J. Perry, M.R.C. Clinical Research Center, Harrow, Great Britain W. Pfleiderer, Fachbereich Chemie, Universität Konstanz, Konstanz, West Germany G.W.E. Plaut, Temple University, School of Medicine, Dept. of Biochemistry, Philadelphia, Penn., U.S.A. M. Poe, Merck Institute for Therapeutic Research, Rahway, New Jersey, U.S.A. H. Rembold, Max-Planck-Institut für Biochemie, Martinsried bei München, West Germany H. Rokos, Fachbereich Chemie, Universität Konstanz, Konstanz, West Germany K. Rokos, Fachbereich Chemie, Universität Konstanz, Konstanz, West Gennany B. Roth, The Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, N.C., U.S.A. P.B. Rowe, Institute of Child Health, Royal Alexandra Hospital for Children, Camper Down, N.S.W., Australia H. Rüdiger, Institut für Pharmazie und Lebensmittelchemie der Universität Würzburg, Würzburg, West Germany H. Sauer, Robert-Bosch-Krankenhaus, Abt. für Hämatologie, Immunologie und Onkologie, Stuttgart, West Germany I. Schwinck, University of Connecticut, Biological Sciences, Storrs, Conn., U.S.A. G. Scrimgeour, University of Toronto, Dept. of Biochemistry, Toronto, Canada P.K. Sengupta, Institut für Organische Chemie, Universität Zürich, Zürich, Switzerland T. Shiota, University of Alabama, The Medical Center, Dept. of Microbiology, Birmingham, Alabama, U.S.A. I. Southon, Fachbereich Chemie, Universität Konstanz, Konstanz, West Germany

XVI A. Stuart, The Wellcome Foundation Ltd., Dartford, Kent, Great Britain H. Sund, Fachbereich Biologie, Universität Konstanz, Konstanz, West Germany E.C. Taylor, Princeton University, Dept. of Chemistry, Princeton, N.J., U.S.A. N. Theobald, Fachbereich Chemie, Universität Konstanz, Konstanz, West Germany M. Viscontini, Institut für Organische Chemie, Universität Zürich, Zürich, Switzerland C. Wagner, Veterans Administration Hospital and Vanderbilt Laboratory, Nashville, Term., U.S.A. S. Waxman, Mount Sinai School of Medicine, New York, N.Y., U.S.A. R. Weber, Institut für Organische Chemie, Universität Zürich, Zürich, Switzerland V.M. Whitehead, Montreal General Hospital, McGill University, Division of Haematology, Montreal, Quebec, Canada J.M. Whiteley, Scipps Clinic & Research Foundation, La Jolla, California, U.S.A. H.C.S. Wood, University of Strathclyde, Dept. of Pure and Applied Chemistry, Glasgow, Scotland, Great Britain R. Wrigglesworth, The Wellcome Foundation, The Wellcome Research Laboratories, Beckenham, Kent., Great Britain

Significant Steps in the Discovery and Application of Pteridines A. Albert

INTRODUCTION Surely, one of the most significant things about pteridines is the keenness of the people who take up this line of research.

That they demand regular

symposia, organize them, and make a success of them is a monument to the vitality of the subject and the people who work in it.

May we never forget

Professor Michel Polonovski who created the first pteridine symposium, in Paris (1952).

For nearly a quarter of a century, these symposia have

grown in size and quality, and I am sure that the present one will be the most successful of all. BEFORE 1940 As is well known, F.G. Hopkins in England isolated fractions rich in pigments from butterfly wings and attempted to determine their nature.

In

his first publication (1889) [1] he described the yellow pigment of the brimstone butterfly.

Six years later, he reported the white pigment from

another Pierid, the cabbage white butterfly [2].

However, other interests

were to claim his attention and the study of insect pigments was allowed to slumber for the next thirty years. Insect Pigments in Munich In 1924, Clemens Schöpf joined Professor H. Wieland as Assistant in>Freiburg University.

A butterfly collector, like the young Hopkins, Schöpf managed

to interest Wieland in the pigments, and they agreed to work on the problem together, organizing school children to catch, and sell them, the butterflies.

Wieland was appointed to the Chemical Laboratories of the Bavarian

Academy of Science in the very next year, and Schöpf went along to continue this work.

Together they extracted and further purified the yellow [3]

and the white [4] pigments which Hopkins had obtained and, coining the name 'pterin1, called then xanthopterin and leucopterin respectively.

However

they met with unexpected difficulties when they tried to determine their structures.

I do not propose to relate, all over again, the early history

2 of their endeavours, already so charmingly told by Clemens Schopf himself in his opening address to the Third Pteridine Symposium in Stuttgart in 1962 [5].

However, I want to call attention to a most paradoxical

situation.

The chemistry of those pyrimidopyrazines, which to-day we call

pteridines, had been very successfully investigated in Berlin from 1894 onwards;

yet in Munich, it was not realized that the butterfly pigments

belonged to this family of compounds until 1940, nearly half a century later. Why did the natural products cause so many difficulties? fitful, because of the seasons, and the material precious. melting points removed an important criterion for purity.

Collection was The lack of Not until 1940

was xanthopterin freed from an impurity that gave a red colour with hydrogen peroxide [6].

The poor solubility in all common solvents diminished ease

of purification and no molecular weights were obtainable.

The resistance

to complete combustion, leading to a nitrogenous coke, produced errors in the analytical results. Particularly misleading was the supposed analogy with purines based on the fact that the pterins and purines gave similar substances upon degradation. All these factors led to a conviction that pterins have a higher molecular weight than is the case.

Thus in 1925, xanthopterin was assigned the

structure of a bipurinyl, namely dihydro-dixanthine (C5H5Nit02)2 (2.)« whereas we know it to be C5H5N5O2.

Xanthopterin ( R = H ) Leucopterin ( R = 0 H ) (1925)

Xanthopterin (1939)

Eight years later, after Metzger had degraded leucopterin to guanidine, it was realized that pterins were richer in nitrogen than had been thought. The latest analytical figures suggested CigHjgNjsOjj for leucopterin, and the diversity of products found after degradation seemed to call for three purine rings per molecule [7].

Xanthopterin was similarly accorded the

formula C19H19N15O7, and the degradative products suggested that both pterins belonged to the general type (3).

(1933)

(1)

In 1936, after a switch from Dumas nitrogen analysis to the Kjeldahl method, and a very painstaking analysis of the much purified barium salt, the formula of xanthopterin was revised to C^gHjgNigOg [8].

However, by 1939

the suggested formula for xanthopterin had been simplified to a symmetrical dimer (2), somewhat similar to that of 1925, essentially a diguanine in place of a dixanthine structure [9]. The year 1940, the second year of the Second World War, seems to have brought a sense of urgency to complete the work.

It also brought the acute,

businesslike mind of Robert Purrmann, one of Wieland's PhD students. Together they reconsidered all the evidence of degradations and also made some more tractable derivatives of leucopterin.

Their conclusion was that

there need be no more than five nitrogen atoms per molecule, and that the choice lay between 2-aminopurine-8-carboxylic acid (4a) or the formula for xanthopterin now known to be correct (4b) [6].

They also recognized, from

a new preparation by simple oxidation, that leucopterin was the mono-oxo derivative of xanthopterin.

4 H

H

(4a)

9 (4b) NH2

(1940)

Xanthopterin

Arguing that purine-8-carboxylic acids lose carbon dioxide w h e n gently heated b u t the pterins did not

[10, 11], Purrmann rejected the purine

alternative for leucopterin and synthesized it by heating 6-oxopyrimidine w i t h oxalic acid

[10].

2,4,5-triamino-

In the same year (1940), he m a d e

xanthopterin from the same pyrimidine and dichloroacetic acid

[11],

correctly establishing the structures of both substances and, in the next year, of another common pterin, isoxanthopterin

[12],

Pteridine Synthesis in B e r l i n The chemistry of pteridine, a name coined by Wieland in 1941

[12] to

describe the nucleus of w h i c h the pterins w e r e derivatives, began in B e r l i n about half a century earlier.

Kiihling, in 1894

[13a] w a n t e d to obtain

the chromophore of tolualloxazine (5), whose constitution w a s well established.

He oxidized the benzene ring to a dicarboxylic acid and

decarboxylated 'alloxazin' in Berlin

[13b] this to the dioxopteridine (6) w h i c h he called

(now known as 'lumazine').

In 1907 Gabriel and Sonn, also

[14], m a d e the same substance by the action of potassium

hypobromlte on pyrazine-2,3-dicarboxamide

Me Tolualloxazine

(D

(_7) .

0

0

H

H

(6)

KOBr

Lumazine

5 In between these two syntheses, there had been only one pteridine publication, but it contained the first example of a general reaction that came to be greatly favoured for the synthesis of pteridines. Isay's condensation [15] of 4,5-diaminopyrimidine 6,7-diphenylpteridine (9).

I refer to Oskar with benzil to give

This was published in 1906.

PhCOCOPh

(1)

W Isay's

Synthesis

The generality of this reaction was soon demonstrated by Sachs and Meyerheim [16] who produced ten more examples of it.

In reporting these, they

mentioned that Traube, when producing 4,5-diaminopyrimidines [17] for his renowned syntheses of purines, had made his students characterize the pyrimidines by condensation with glyoxal, but this was mentioned only in their dissertations.

Sachs and Meyerheim called their products 'azin-

purine', and no connexion with the butterfly pigments was suspected. The question must now be asked:

How did the Berlin chemists manage to

proceed with such speed and confidence in what was later to prove a difficult field?

In the first place, they knew the composition of their starting

materials, and were able to obtain the pteridines from them in one step for which good analogies existed in related series.

Nor were they worried by

quantities, for the starting materials were reasonably well available.

It

is, perhaps, surprising that they were not troubled by the lack of meltingpoints or by the current (macro) analytical methods.

However they were

proceeding in an atmosphere created by Emil Fischer who did most of his celebrated purine work in Berlin, between 1882 and 1900, and who must have encountered similar problems in that series. Yet another example of pteridine synthesis was published before the constitution of the wing pigments was worked out in Munich.

This was a third

method for preparing lumazine (from 4,5-diamino-2,6-dioxopyrimidine and glyoxal), achieved by Kuhn and Cook (in Heidelberg) [18] who recognized it as a degradation product of riboflavine.

6 FROM THE SYNTHESIS OF XANTHOPTERIN TO FOLIC ACID Once the constitution of the principal butterfly pigments had been determined, and they had been found also in wasps and bees, many laboratories took up the search for pteridines in Nature.

Paper chromatography, which

had only just arrived on the scene, was pressed into service in conjunction with the traditional fluorescence lamp (365 nm).

For the most part, this

search was not very efficient because many pteridines do not fluoresce, and many substances which fluoresced turned out not to be pteridines.

Moreover,

the zoologists who undertook most of this work seldom isolated enough of a new material to have its chemical structure determined.

In the period

1940-1950, many insects, silkworms, amphibia, and fish were looked at in this way.

France, Switzerland, and Japan were early arrivals in this

field of activity which later spread all over the world.

Some pteridines

were found to be essential coenzymes and others developmental hormones, whereas still others (including, it would seem, xanthopterin and leucopterin) are breakdown products of these, accumulated only through a genetic error in metabolism.

Too many of the developmental hormones are still recognized

only by their Rp and fluorescence colour because their chemical nature is not known.

Closer links between biologists and chemists could lead to

the synthesis of these hormones with probably startling results.

This

work should prove more fruitful than the investigation of pigments of no known biological function. FOLIC ACID AND ALL THAT IT LED TO The story of folic acid illustrates the widespread (and beneficial) consequences that can fan out from the isolation of a biologically active pteridine and determination of its structure.

About 1940, fractions

isolated from green leaves, yeast, and liver were found to have anti-anaemic properties and to be growth factors for some micro-organisms.

Thus

Mitchell's 'folic acid', isolated from spinach [19], promoted the growth of L. aasei and some other bacteria.

In 1946, the structure of folic acid

was found by degradation and synthesis [20].

It occurs naturally as the

dihydro-derivative (10). The function of folic acid is to insert a single carbon atom wherever this is required in biosynthesis.

To .this end, dihydrofolic acid is reduced,

7 HO2CCH2 CHZ

(10)

0

H 0 2 C CH NH

C

0

~ ^

_

N

H

_

C

H

2

Y

N

T |

I

H

^ S R ^ N ^ N H g H

Dihydro-folic acid

by the enzyme dihydeofolate

reductase,

to tetrahydrofolic acid which forms

the required coenzyme by uniting with an appropriate one-carbon fragment. This fragment is inserted into the molecule undergoing biosynthesis and the coenzyme is reconstituted in a cycle of oxidation and reduction which proceeds in one-electron steps.

The four principal kinds of coenzyme are shown

in Scheme 1.

THFA

PURINES

formate

DHFA •

• THFA

/?

(5)'*(10)" M e t h e n y l

-Methylene - THFA

tf^-Methyl

Scheme 1.

PURINES

THFA

•THYMIDYLIC ACID

- THFA

METHIONINE

Biosynthetic functions of folic acid coenzymes [DHFA and THFA: di- (and tetra-) hydrofolic acid]

Thus in the biosynthesis of purines, all of which proceeds through inosinic acid (12), the methenyl

coenzyme is needed to insert C-8.

This insertion

is actually performed on glycinamide ribotide which becomes the ribotide of 4-aminoimidazole-5-carboxamide ('AIC') (11).

The forrnyl

coenzyme is then

used to insert C-2 into the purine (12), namely by formylation of the ribotide of AIC.

8

(li)

Ü2)

Ribose-P04 AlC

Inosinic

acid

o O

(13)

Ri

NH

H

Uracil ( R = H ) , T h y m i n e (R= Me) Deprivation of folic acid, either directly or through use of an antagonist, leads to death.

In some organisms death occurs through failure to

synthesize purines as fast as they are lost, in others through inability to produce enough thymine (13) from uracil to replace lost DNA. As soon as it was seen that p-aminobenzoic acid was part of the folic acid molecule (and it does not occur elsewhere in Nature) it was realized that the sulphonamide drugs owed their antibacterial action to interference with the synthesis of folic acid.

The selectivity of these drugs depends on two

facts, that mammals do not synthesize their own folic acid but absorb it from the diet, and that hardly any pathogenic bacteria can absorb folic acid but most of them must synthesize their own.

The antibacterial

sulphonamides inhibit the enzyme that condenses p-aminobenzoic acid with 2-amino-4-oxo-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. enzyme (dihyd.rofolate

synthetase)

This

has been isolated and purified by

G.M. Brown [21] and shown to be reversibly inhibited in this way.

In

addition, some sulphonamides are accepted by the enzyme and made into rogue folic acids. Folic acid is an indispensable pro-vitamin for man, and lack of it quickly causes macrocytic anaemia and gastro-intestinal disorders.

During pregnancy

it is commonly prescribed to prevent development of the typical anaemic state.

Hence it was with some trepidation that chemists began to make

9 metabolite analogues, based on the pteridine nucleus, as possible drugs. Nevertheless many very valuable remedies have been obtained in this way. Methotrexate (14), introduced into the clinic in 1958, has proved to be an excellent and reliable drug in certain forms of cancer.

This substance

differs from folic acid in two particulars, the oxo-function in the 4positioft has been replaced by an amino-group, and the hydrogen atom in the 10-position by a methyl-group.

Methotrexate, alone or in combination with

other inhibitors of DNA synthesis, provides the standard treatment for the leukaemia of young adults [22, 23].

In addition, methotrexate provides

a rapid and complete cure for two other, otherwise fatal, forms of cancer: choriocarcinoma (a fast-growing tumour of pregnancy) and Burkitt's lymphoma (a solid tumour of the jaw affecting many children in Africa) [23].

It is also used in severe cases of psoriasis.

H0 2 C CH 2

OA)

!..

CH 3

^

Methotrexate

Methotrexate acts by inhibiting dihydrofolate

reduota.se-.

50 per cent

-9

inhibition is effected by a 10 M concentration of this drug which has hardly any effect on any other enzyme.

It is bound to the enzyme about

101* times more tightly than the substrate, surely the least reversible of all reversible inhibitors.

Unfortunately it has little toxicity for most

bacteria and protozoa, because it can no more be taken up by these organisms than folic acid can.

10 To obtain antagonists of dihydrofolate reductase which could penetrate bacteria and protozoa by simple diffusion, G.H. Hitchings and his colleagues began the systematic simplification of the molecule of methotrexate.

They

found that quite simple 2,4-diaminopyrimidines, provided with aryl substituents, can penetrate into these organisms where they exert a strong antagonistic action on this enzyme [24].

Pyrimethamine (15), discovered

in this way, has become the most widely used of all prophylactics against malaria.

It is now known that an enzyme performing a certain function in

a parasite can be chemically different from one performing the same function in the host.

This is known as the principle of analogous enzymes.

Pyrimethamine is highly selective in this way:

it inhibits the enzyme from

the malarial parasite about 2000 times more strongly than any analogous mammalian enzyme.

Similar selectivity is shown against bacteria by

trimethoprim (16) (see Table 1).

It will be realized that sulphonamides

and trimethoprim block two consecutive stages in the synthesis of the tetrahydrofolate coenzymes.

It has proved very advantageous to give them to

patients simultaneously, thus achieving a large synergistic effect through what is known as 'sequential blocking'.

This combination is now much

prescribed for oral treatment of bronchitis and in infections of the bowel, kidney, and bladder. (Parenthetically speaking, so remarkably do anti-folic drugs distinguish between host-parasite metabolic pathways, and also analogous enzymes, and so remarkable is their action in sequential blocking, that any discussion on these three topics is always illustrated by reference to the antifolies.) Table 1. Mammal

Inhibition of (isolated) Dihydrofolate Reductase by Trimethoprim a Trimethoprim'

Bacterium

a Trimethoprim'

Man

30,000

Esoherichia eoli

0.5

Rat

26,000

Staphylococcus aureus

1.5

Rabbit

37,000

Proteus vulgaris

0.4

a

Concentration (x 108M) causing 50% inhibition

11 BIOPTERIN AND RIBOFLAVINE The participation of pteridines in essential metabolism is by no means restricted to folic acid derivatives.

In 1956, Patterson and colleagues

isolated biopterin (17) from human urine and synthesized it in the same year [25].

This work was done in the Lederle Laboratories of the American

Cyanamid Co. where the degradation and synthesis of folic acid had taken place ten years earlier [20].

It was then found, mainly by S. Kaufman,

that 5,6,7,8-tetrahydrobiopterin is the co-factor for a set of enzymes that use molecular oxygen to introduce a hydroxy-group into (respectively) phenylalanine, tyrosine, and tryptophan [26] and possibly, too, in the hydroxylation of steroids and the formation of melanin.

Biopterin is

present in the royal jelly of the honey bee, but is absent from the workers' food [27].

Several syntheses of-biopterin have been described, but it is

still very expensive. freely.

Were it more abundant, it could be used more

Abnormalities in phenylalanine metabolism lead to mental sub-

normality, and failure to hydroxylate tyrosine would prevent synthesis of all the catecholamines;

there may be clinical uses for biopterin to be

developed here, if it were more abundant.

0

Biopterin Contrary to early assumptions, an amino-group is not essential for biological activity in the pteridine series.

The biosynthesis of the

vitamin riboflavine takes place [28] via the pteridine 6,7-dimethyl-8ribityl-2,4-dioxopteridine (18) first isolated from the riboflavineproducing mould Eremotheoivm

asKbyi

[29].

Other 2-oxopteridines, such as

6-hydroxymethyl-lumazine, have been found in Nature [30].

12 0 Me Me I CH,

(iâ)

H

Step in biosynthesis of

Riboflavine

BIOSYNTHESIS The biosynthesis of folic acid, like that of riboflavine, begins with a purine, in this case guanosine triphosphate, which becomes metabolized to the pyrimidine (19).

Ring-closure gives dihydro-neopterin triphosphate

(20) which is surprisingly like biopterin (17) but has the opposite optical configuration (D instead of L).

The enzyme hydvoneopterin aldolase then

splits out glycolllc aldehyde, leading to the pteridine (21) which is readily built into dihydrofolic acid (10) [31]. It is a fascinating thought that the folic coenzymes which regulate the synthesis of purines are themselves made from a purine, and here we may see a process of self regulation that may get out of hand in malignant conditions [32].

H

0

(20)

U£) HO

H

OH

( T = Triphosphate group)

Dihydro-neopterin triphosphate

Degraded G T P

0

(21)

H (P-pyrophosphate Stages in biosynthesis

group)

of Folic Acid

13 THE SPREAD O F INTEREST IN SYNTHESIS The synthesis of pteridines, w h i c h so far had b e e n practised only in Germany, spread to many other countries about 1946, the year in w h i c h the structure and functions of folic acid w e r e disclosed.

Some groups took up pteridine

synthesis for the sheer fascination of its difficult and paradoxical chemistry, others to prepare candidate drugs for biological tests, others to help natural product workers, and yet others to correlate structure w i t h properties, whether physical, biochemical, or physiological. Our A u s t r a l i a n group, quartered at that time in London, entered the field in 1949, publishing first in 1951. in the field.

In Germany, the m o s t active centre was, by then, that of

Tschesche and Korte in Hamburg. England:

W e soon got to know the principal workers

Several active groups had sprung up in

Boon and Jones in Manchester

(in 1948 Jones discovered the parent

substance, pteridine), Ramage in Huddersfield (later in Salford), Forrest and Walker in London, and Timmis in the L o n d o n area.

In Paris, M i c h e l

Polonovski, who had entered the field by isolating xanthopterin from a crab, soon built up a synthetic group that included Pesson.

In the U.S.A.,

apart from the Cyanamid Co., there was the Cornell University group of Taylor, Cain, and Mallette who first published in 1946. Karrer w a s active in the University of Zurich. strengthened further.

In Switzerland,

In the 1950's, the situation

Pfleiderer's first publication (1954) from Stuttgart

signalled the beginning of a strong school, w h o s e hospitality w e are n o w enjoying in Konstanz.

Taylor transferred to Urbana (Illinois) then to

Princeton, founding another strong school, as did Viscontini w h o took over from Karrer in Zurich.

Two m e n who left our A u s t r a l i a n group for n e w

universities in Britain have remained very active in pteridine chemistry: W o o d at Strathclyde and Clark at Salford.

Lister has rejoined D.J. B r o w n

in Canberra, but is now a purine specialist. The Japanese Contribution I w a n t to add a special note o n the Japanese school.

This became quite

strong early, through the inclusion of Egami, at that time Japan's m o s t illustrious biochemist, in the first Pteridine Symposium in Paris.

Egami

w a s a graduate of Strassbourg University and communicated freely in F r e n c h and English.

Japanese pteridine chemistry has largely concentrated o n

14

economic products such as fermented foods and drink, and the silkworm and fish industries.

It seems to find readier government support than in many

other countries.

The fourth Pteridine Symposium in Toba (1969) enabled

many Western scientists to meet their opposite numbers in Japan.

Our

Australian group had some very happy reunions, for a steady stream of Japanese graduate students and postdoctorals have worked with us in Canberra Matsuura, now Professor at Nagoya, was the first of these. The Australian Programme I hope you will allow me to say a few words about our own aims.

These were

to correlate structure with physical properties in order to explain the many anomalies in the pteridine series and to provide a rational basis of pteridine chemistry for workers in biochemistry and the medical sciences. To this end, we produced a great many mono-substituted pteridines, at that time an unknown category of substance, and compared them with the parent substance and with more highly substituted pteridines.

We found that the

troublesome properties that had bedevilled the Munich work increased in proportion as amino- and oxo-groups were inserted into the nucleus. Pteridine itself is low-melting, volatile, very soluble in water and in organic solvents, and remarkably easily broken down by dilute acid and alkali.

These properties persisted, although clearly diminished, when a

single amino- or oxo-group was inserted, whereas the presence of three or four such groups, as in the wing pigments, produced substances that would not melt, dissolve, or even burn completely.

Table 2.

Solubilities of Pteridines in Water at 20°

Pteridine Unsubstituted 2-Amino 2-Dimethylamino 7-Oxo-

1 part in: 7 1350 3 900

O-Methyl derivative

50

/'/-Methyl derivative

50

The anomalies of solubility are illustrated in Table 2.

Whereas the parent

substance, pteridine, is soluble in 7 parts of cold water, the introduction of an amino- or an oxo- ("hydroxy-") group makes it far less soluble.

Each

further insertion of either of these groups still further reduces solubility (see Table 3), so that tetraoxopteridine requires 58,000 and leucopterin 750,000 parts [33]. purine series.

Similar effects had been observed by Fischer in the

We traced them to hydrogen bonding, noting that solubility

returns upon either N- or 0-methylation (see Table 2).

Our explanation was

that the ir-deficient character [34] of heteroaromatic rings increases with every doubly-bound nitrogen atom.

This results in an accumulation of

electrons by these atoms which can out-compete the oxygen of water for union with bondable hydrogen atoms.

This effect not only reduces aqueous

solubility but, by increasing crystal lattice energy, raises the meltingpoint and increases resistance to combustion.

Table 3.

Solubilities of Pteridines in Water at 20°

Pteridine Unsubstituted

1 part in: 7

2-0xo-

600

4-0xo-

200

7-0xo4,7-dioxo-

900 4000

2,4,7-trioxo-

12000

2,4,6,7-tetraoxo-

58000

The highly ir-deficient character of the pteridine ring prevents electrophilic substitution but makes it particularly vulnerable to nucleophilic attack.

Thus, boiling dilute acid (and alkali too) largely destroys the

parent substance in an hour, an effect which can be countered by the cumulative introduction of electron-releasing groups, such as -NH2 and =0 (the latter with its associated NH constitutes an intracyclic amide group) This is illustrated in Table 4.

16 Table 4.

Decomposition of Pteridines by Boiling N-I^SO^

Pteridine

Decomposition in 1 hour

Unsubstituted

74 %

2-0xo-

55

4-0xo-

60

2,4-dioxo-

(lumazine)

6

6,7-dioxo

7

4,6,7-trioxo-

0

2,4,6,7-tetraoxo-

0

W e determined many ionization constants, having h a d m u c h experience in applying the appropriate techniques to heterocycles

[35], and w e combined

the results w i t h evidence from ultraviolet spectra to determine the principal tautomeric state of the m o n o - a m i n o - and 'hydroxy 1 -pteridines. 0-

methyl-derivatives w e r e u s e d as controls

[36].

Both N-

and

This w o r k w a s extended

by Pfleiderer to polysubstituted pteridines. By potentiometric titration in the presence of heavy m e t a l cations, 4-oxopteridines were found to b e active chelating agents

[37].

COVALENT HYDRATION In 1952, w e discovered the first instance of 'covalent h y d r a t i o n 1 , w h i c h has turned out to be a w i d e s p r e a d phenomenon in the pteridine series a n d in related polyaza-heteroaromatic compounds.

It is essentially the addition

of water across a C=N double bond to give a secondary or tertiary alcohol. The nucleus of pteridine is n o t truly aromatic because the Ti-double layer has been depleted by several doubly-bound nitrogen atoms.

Consequently

some of the C=N bonds take o n the character of isolated double bonds (as is apparent in X-ray diffraction reagents

analysis)

and avidly add nucleophilic

[38].

The phenomenon can b e detected from the ionization constants w h i c h show considerable strengthening of basic, and weakening of acidic, properties because of the saturation of the double bond.

Similarly,

ultraviolet

17 spectra show changes characteristic of saturation of a double bond.

Nuclear

magnetic resonance, too, can detect covalent hydration by the large upfield shift of the proton in a CH-group at the site of hydration

[38].

Essentially

the double-bond polarizes, as in (22) , and attracts a water molecule which adds as in (23);

finally the negative charge on the ring-nitrogen atom is

neutralized by protonation.

(22)

(23)

Steps in h y d r a t i o n of

Pteridine

Our first example was discovered during the potentiometric titration of 6-oxopteridine with alkali followed by back-titration with acid, which traced out the hysteresis loop shown in Fig.l

[39].

Comparison of the

spectrum of 6-oxopteridine (neutral species) with those of its 2-, 4-, and 7-methyl derivatives

[40a] showed that only the latter had the sharp,

relatively high wave-length peak characteristic of 4- and 7-oxopteridine, whereas the other three spectra showed only a little of this peak but a great deal of another, at shorter wave-length and similar to that of 7,8dihydro-6-oxopteridine

(see Fig.2).

As solid '6-oxopteridine' is strongly

bound to water in a 1:1 ratio, we assigned it the constitution 7-hydroxy-6-oxopteridine

[40].

7,8-dihydro-

This is a weak acid (p^ a 9.7) which, during

titration w i t h alkali gives the anhydrous anion;

back-titration with acid

furnishes the anhydrous neutral species, namely true 6-oxopteridine, which is a much stronger acid of p K a 3.7. hydrated;

However the latter rapidly becomes

the hysteresis loop can be retraced over and over again.

Later

we found that most covalent hydrations went too fast for hysteresis to be detected in this way;

yet both hydrated and anhydrous spectra could be

recorded in 'rapid reaction apparatus' and from them ionization constants were often determined for both the hydrated pair of species and for the anhydrous pair [41].

In the above example, the methyl-group of 7-methyl-

6-oxopteridine is hindering hydration by both steric and electronic effects (see below).

18

pH

N

N

PTERID-6-0NE

/ /

i

/ t

I Eq HCl

/

/

i

/

i is" >/

t

Starting Point

t

I Eq NaOH

Fig.l.

Potentiometrie titration of 6-oxopteridine

Fig.2.

Ultraviolet spectra of the neutral species (at pH 5.5) of (A) 6-oxopteridine and (B) its 7-methyl-derivative

Fig.3.

Ultraviolet spectra of the neutral species (at p H 4.0) of (A) xanthopterin and (B) its 7-methyl-derivative

19 That xanthopterin, in neutral solution, consists of an equilibrium mixture of two substances was noticed in 1950 by Schou (42].

He attributed this to

tautomerism, but we found that it is actually the equilibrium mixture of about equal parts of anhydrous xanthopterin and its covalent hydrate. the anhydrous form is fluorescent.

Only

On paper chromatography, pure xantho-

pterin always gives two spots, of which the non-fluorescent one can be clearly seen under a 254 nm ultraviolet lamp.

The pure substance in each

spot, when eluted and re-applied to paper, develops both the spots.

Fig.3

shows the ultraviolet spectrum of the equilibrium mixture of (A) xanthopterin and the spectrum of (B) its 7-methyl homologue which is not hydrated.

The

hydrate of xanthopterin is evidently absorbing heavily around 320 nm.

It

will be recalled that the Munich school had 7-methylxanthopterin as a contaminant in most of their xanthopterin samples right up to 1940.

Apart

from this difficulty, a comparison of the spectra would not have revealed that the two substances were simple homologues.

Further, the hydrated and

the anhydrous forms of xanthopterin may have undergone degradation in two different ways, giving rise to the multiplicity of products of which they complained. Table 5 presents the ionization and ultraviolet data for 2-aminopteridine and two of its homologues.

The steady loss of basic strength (quite

contrary to the usual rule) as methyl-groups are inserted, signals the progressive blocking of hydration which takes place mainly in the 3,4position, but also in the 7,8-position.

A study of the ultraviolet spectra

shows that 2-aminopteridine is completely hydrated as the cation;

as

hydration is prevented, a new strong peak at longer wave-lengths appears. The neutral species, however, is not hydrated. Table 5.

Ionisation and ultraviolet absorption of 2-aminopteridine and homologues HO^-H

H SPECTRA 2-AMINOPTERIDINE

fKa

NEUT M O L X max

1 CATION loq £

A max

1

3 92. 3 87

4 3

2 2 5 . 370rrv 232. 3 0 2

4-Melliyl

2 8

2 2 5 . 367

217. 307, 3 4 6

4 7-Dlmethyl

2 6

228. 363

218

(unsubst)

347

4 2 7 3 7 2 . 3 71 4 27

4 02

20

Here w e m i g h t pause a n d ask, 'What are the factors responsible for covalent hydration?'

First of all, the m o l e c u l e m u s t b e sufficiently

electron

depleted, either by the presence of more than one doubly-bound nitrogen atom, or by strongly electron-attracting substituents such as - N O 2 , or -SC^Me. These circumstances encourage nucleophilic attack, by water molecules in this case.

But this provision is n o t enough to give an instrumentally

strable amount of hydration;

demon-

it is necessary in addition to have resonance

stabilization of the hydrate.

A n example of this type of stabilizing

influence is the amidinium resonance, well known to operate in the cations (but not in the neutral species) of amidines, thus making them strong bases. Scheme 2 shows how the cation (but not the neutral species) of quinazoline is stabilized by an amidinium resonance, a n d the similar (if m o r e complex) behaviour of the pteridine cation will be discussed later.

By a different

type of resonance (the amide type), 6-, and also 2-, oxopteridine hydrates are stabilized as the neutral species, but n o t as the anion.

The hydrate

of pteridine forms a n anion, just as amidines do, and this is stabilized by resonance.

Neutral pteridine has only about 20% of hydrate at equilibrium

[41]. RESONANCES 1

The A M I D I N I U M NH,

THAT

„•

^CH

HYDRATIONS

NH,

NH, II CH

* CH RN H

RN

Example

Quinozoline

H

RN H

Hrfdrote

OH

NH N n m (unstable)

Scheme 2.

STABILIZE

RESONANCE

H

OH

H+

^»-/"'N^

V H^y

cation(stoble)

Example of a hydration-stabilizing

resonance

Additional evidence of the covalent character of these covalently

solvated

products comes from mass spectrography (where hydrated and anhydrous species degrade very differently)

[43] a n d X-ray diffraction studies

[44].

After some time, the site of hydration of a m o l e c u l e can shift

[45].

The

water molecule first, under kinetic control, adds to a site w i t h adequate affinity and little energy barrier.

After an interval of time, it m a y b e

found that the water molecule has accumulated at a different site in the

21

same molecule, provided that its affinity is greater and the energy barrier greater but not insurmountable (it is now under thermodynamic control). example can be seen in Fig.4.

An

The cation of pteridine, which rapidly

hydrates in the 3,4-position as soon as it is formed (by dissolving pteridine in dilute acid), has a complex multiplet at about x 1.3 representing protons on the 2,4,and 6-positions and a sharp upfield peak representing CH in the 4-position, which now provides an aliphatic setting.

However, with a half

time of 20 minutes, this picture changes to a new equilibrium where singlet peaks, downfield, represent the 2- and 4- protons, whereas those on 6- and 7- unite to a complex multiplet signifying dihydration across the 5,6- and 7,8- double bonds.

Thus the lower graph in Fig.4 shows both the kinetically

and the thermodynamically controlled addition of water to the same cation [46]. P T E R I D I N E CATIONS

0H1

TO

Fig. 4.

I

t. • lOmm(lt)

The kinetically and the thermodynamically controlled addition of water to the pteridine cation at pH 1.

Table 6 presents some examples of the influence of steric and electronic effects on covalent hydration of pteridines;

the examples of 4-trifluoro-

methylpteridine [47] and 4-ethoxycarbonylpteridine [48] add variety to the types already discussed.

The hydrated product (4-ethoxycarbonyl-5,6,7,8-

tetrahydro-6,7-dihydroxypteridine) of the last named substance can be obtained in both a£s and trans geometrical forms [48].

22

Table 6.

Nucleophilic Attack of Water Molecules on Pteridine Cations Effect on attack, at 4-

Site of hydration

Steric

Electronic

Initial

H-

nil

nil

3,4

Me-

hinders

hinders

5,6-7,8

5,6-7,8

F3C-

hinders

attracts

5,6-7,8

3,4

ETO.CO-

prevents

attracts

5,6-7,8

5,6-7,8

Substituent in 4-position

Final 5,6-7,8

Unusual types of hydration have been encountered and studied in the 8-alkyllumazine and -pterin series (these models relate to intermediates in the biosynthesis of riboflavine).

In these substances, the alkylation of a

'borrowed hydrogen atom' in the 8-position has led to strained, quinonoid forms, highly subject to hydration [49]. Other Nucleophilic Additions We found that all mono-oxopteridines, whether they added water or not, underwent nucleophilic addition reactions with sodium bisulphite, Michael reagents, barbituric acids, and dimedone [40a, 50].

Such mono-amino and

mono-thio pteridines as were examined did this also [51]. look into the addition reactions of the parent. Scheme 3, these fell into three classes.

This led us to

As can be seen from

Many examples of simple addition

across the 3,4-double bond were found, sometimes preceding transfer to the pyrazine ring.

In another pattern, exemplified by cyanoacetamide, the

pyrimidine ring opened, expelled a nitrogen atom, and closed again.

A third

pattern, as shown by acetylacetone, led to 1:1 addition across the 6,7-bond [52],

MaO

M«0

Scheme 3.

H

(Albert I fllzuno. 1*71, 1973)

Addition reactions of pteridine

23 Pteridines also add nucleophilically to one another, either directly as in the purple dimer of 2-oxopteridine (24), or through a CH2-bridge, as in the dimer (25) (R = H) of 4-methylpteridine [53]. Evans and Wolfenden showed that pteridine is changed by the enzyme adenosine deaminase to the Z-aeworotatory form of the hydrate 3,4-dihydro-4-hydroxypteridine.

They concluded from this that covalent hydration is the first

stage in the deamination of adenosine [54].

(24)

(25)

APPLICATIONS OF PTERIDINES Several medical applications of pteridines have already been mentioned.

One

of a different kind is the use of the diuretic triamterene (26) (2,4,7diamino-6-phenylpteridine) and the slightly less active 2,4-diamino-6,7dimethylpteridine.

These act, in the distal tubule of the kidney, on the

system which exchanges Na~*" for K"*" [55] .

Some recent Boehringer patents

suggest that quite simple pterid-4-ones have diuretic properties.

Ph^N. (26)

L

JL is.

A R

(27)

H

(R= H y d r o x y a l k y l ) Triamterene

( R ' = d o . , Me or H ) Nippon Zoki Analgesics

Glycosides of pteridines, such as 8-D-ribofuranosyl-lumazine, made by Pfleiderer and associates, have been found to have viricidal and bactericidal properties [56],

A search for inhibitors of dihydrofolate reductase

in the bacterium that causes leprosy has found good activity (but poor cell permeability) in 2,4-diamino-6-isobutylpteridine [57].

The enzyme

24 6-hydroxymethyl-7

¡S-dihydropterin

pyrophosphokinase, which is necessary for

the biosynthesis of dihydrofolic acid (see foregoing), can be inhibited by substances recently synthesized by Wood and colleagues, such as 2-amino-7,7diethyl-6-hydroxymethylpterid-4-one and 6-formyl-7,7-dimethyl-7,8-dihydropterin which have proved useful for treating wounds after operations

[58].

A recent Japanese patent claims pteridines of the type (27) as useful analgesics and inhibitors of inflamination [59]. In photosynthesis, there is some evidence that an unknown pteridine is (at a very low potential) the primary acceptor of the electrons produced from absorption of the light quanta from photochemical energy

[60].

CONCLUSION The time has been too short to deal adequately with all the significant steps and there may even be several that I have overlooked.

This matters

less, because of the great feast of new contributions which we are to hear in our five days in Konstanz, out of which much of significance will surely emerge.

REFERENCES 1.

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Hopkins, F.G., Philosoph. Trans. Roy. Soc. (London), Ser. B, 186, 661-682 (1895).

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Wieland, H., and Schöpf, C., Ber. Deut. Chem. Ges., _58, 2178-2183 (1925).

4.

Schöpf, C., and Wieland, H., Ber. Deut. Chem. Ges., 59, 2067-2072 (1926).

5.

Schöpf, C., in 'Pteridine Chemistry' (Proceedings of the Third International Symposium), pp.3-14, Pergamon Press, Oxford (1964).

6.

Wieland, H., and Purrmann, R., Liebigs Ann. Chem., 544, 163-182 (1940).

7.

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8.

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25 14.

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16.

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20.

Angier, R.B., Boothe, J.H., Hutchings, B.L., Mowat, J.H., Semb, J., Stokstad, E.L.R., SubbaRow, Y., Waller, C.W., Cosulich, D.B., Fahrenbach, M.J., Hultquist, M.E., Kuh, E., Northey, E.H., Seeger, D.R., Sickels, J.P., and Smith, J.M., Science, 103, 667-669 (1946).

21.

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22.

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23.

Brule, G., Eckhardt, S.J., Hall, T.C., and Winkler, A., 'Drug Therapy of Cancer', pp.163, World Health Organization, Geneva, 1973.

24.

Burchall, J.J., and Hitchings, G.H., Molec. Pharmacol., _1, 126-136 (1965); Hitchings, G.H., and Burchall, J.J., Advances Enzymol., 27. 417-468 (1965).

25.

Patterson, E.L., Milstrey, R., and Stokstad, E.L.R., J. Amer. Chem. Soc., 78, 5868-5871 (1956).

26.

Kaufman, S., in 'Pteridine Chemistry' (Proceedings of the Third International Symposium), pp.307-326, Pergamon Press, Oxford (1964); Ciba Foundation Symposium, 22, 85-115 (1974).

27.

Butenandt, A., and Rembold, H., Hoppe-Seyler's Zeit, physiol. Chem., 311, 79-83 (1958).

28.

Plaut, G.W.E., J. Biol. Chem., 238, 2225-2243 (1963).

29.

Masuda, T., Pharmac. Bull. (Tokyo), A, 71-72 (1956) (per Chem. Abs., 1957, ,51, 2931).

30.

Sugiura, K., and Goto, M., Nippon Kagaku Zasshi, 87, 623-624 (1966).

31.

Shiota, T., Jackson, R., and Baugh, C.M., in 'Chemistry and Biology of Pteridines' (Proceedings of the Fourth International Symposium), pp.265-279; Brown, G.M., idem., pp.243-264, International Academic Printing Co., Tokyo (1970).

32.

Albert, A., Biochem. J., 65, 124-127 (1957).

33.

Albert, A., Brown, D.J., and Cheeseman, G., J. Chem. Soc., 4219-4232 (1952); Albert, A., Lister, J.H., and Pedersen, C., J. Chem. Soc., 4621-4628 (1956).

34.

Albert, A., 'Heterocyclic Chemistry', pp.547, 2nd Edn., Athlone Press, London (1968).

35.

Albert, A., Goldacre, R., and Phillips, J., J. Chem. Soc., 2240-2249 (1948); Albert, A., and Phillips, J.N., J. Chem. Soc., 1294-1304 (1956); Albert, A., and Serjeant, E.P., 'The Determination of Ionization Constants', pp.115, 2nd Edn., Chapman & Hall, London (1971).

26

36.

Brown, D.J., and Mason, S.F., J. Chem. Soc., 3443-3453 (1956).

37.

Albert, A., Biochem. J., 54, 646-654 (1953).

38.

Albert, A., Angew. Chem., _79, 913-922 (1967); Edn. (English), 6, 919-928 (1967).

39.

Albert, A., Brown, D.J., and Cheeseman, G., J. Chem. Soc., 1620-1630 (1952).

40.

(a) Albert, A., and Reich, F., J. Chem. Soc., 127-135 (1961);

41.

Perrin, D.D., Adv. Hetero. Chem., _4, 43-74 (1965).

Angew. Chem. Internat.

(b) Brown, D.J., and Mason, S.F., J. Chem. Soc., 3443-3453 (1956). 42.

Schou, M., Arch. Biochem., 28, 10-29 (1950).

43.

Clark, J., and Cunliffe, A., Org. Mass Spectrom., _7> 737-752 (1973).

44.

Batterham, T.J., and Wunderlich, J.A., J. Chem. Soc. (B), 489-494 (1969).

45. 46.

Albert, A., Inoue, Y., and Perrin, D.D., J. Chem. Soc., 5151-5156 (1963). Albert, A., Batterham, T.J., and McCormack, J.J., J. Chem. Soc. Q5), 1105-1109 (1966).

47.

Clark, J., and Pendergast, W., J. Chem. Soc. (£), 1751-1754 (1969).

48.

Clark. J., J. Chem. Soc. (C), 313-317 (1968).

49.

Pfleiderer, W., Bunting, J.W., Perrin, D.D., and NUbel, G., Chem. Ber., 99, 3503-3523 (1966); Pfleiderer, W., Bunting, J.W., Perrin, D.D., and Nubel, G., Chem. Ber., 101, 1072-1088 (1968).

50.

Albert, A., and Howell, C.F., J. Chem. Soc., 1591-1596 (1962); Albert, A., and McCormack, J.J., J. Chem. Soc., 6930-6934 (1965); Albert, A., and McCormack, J.J., J. Chem. Soc. Perk.I, 2630-2632 (1973).

51.

Albert, A., and McCormack, J.J., J. Chem. Soc. (£), 63-68 (1968); 1117-1120 (1966).

52.

Albert, A., and Mizuno, H., J. Chem. Soc. (B), 2423-2427 (1971); J. Chem. Soc. Perk.I, 1615-1619 (1973); 1974-1980 (1973).

53.

Albert, A., and Reich, F., J. Chem. Soc., 1370-1373 (1960); and Yamamoto, J. Chem. Soc. (C), 1181-1187 (1968).

54.

Evans, B., and Wolfenden, R., J. Amer. Chem. Soc., 94, 5902-5903 (1972); Biochemistry, 12, 392-398 (1973).

55.

Wiebelhaus, V.D., Weinstock, J., Maass, A.R., Brennan, F.T., Sosnowski, G., and Larsen, T.J., J. Pharmacol., _149, 397-403 (1965).

56.

Pfleiderer, W. (to Ciba-Geigy Corp.), U.S. Pat. 3792036 (1974) (per Chem. Abs., 1974, 80, 121292).

57.

DeGraw, J., Brown, V., Colwell, W., and Morrison, N., J. Med. Chem., 11_, 144-146 (1974).

58.

Wood, H.C.S., and Stirling, I. (to Wellcome Foundation), German Pat. 2338787 (1974) (per Chem. Abs. 1974, 80, 108579); Wood, H.C.S., Whittaker, N., Stirling, I., and Ohta, K., German Pat. 2404593 (1974).

59.

Takino, M., Kurosaki, T., and Odaka, M., German Pat. 2346122 (1974) (per Chem. Abs., 1974, jU, 4220).

60.

Fuller, R.C., Kidder, G.W., Nugent, N.A., Dewey, V.C., and Rigopoulos, N., Photochem. Photobiol., 359-371 (1971).

Albert, A.,

Specific Inhibition of Dihydrofolate Biosynthesis A New Approach to Chemotherapy H.C.S. Wood

INTRODUCTION The essence of chemotherapy is the differential effect whereby the drug is more detrimental to the parasite than to the host.

Selective toxicity is therefore the key to the design

of successful chemotherapeutic agents.

This simple fact is

often ignored in the planning of research programmes. Traditional methods for the discovery of chemotherapeutic agents can best be summed up in the phrase empiricism'.

'enlightened

Success depends upon the availability of new

compounds, efficient biological screening

facilities,

appropriate feed-back from biologists to chemists, and hence to chemical modification of the candidate drug.

Surprisingly,

such trial and error methods work, and the majority of chemotherapeutic agents which are available today have been discovered in this way.

The vast allocation of resources of

men and materials for this approach make it necessary to question this method more than ever at the present

time.

The present essay records an attempt to investigate

the

rational design of antibacterial agents based on studies of the biosynthesis of dihydrofolate.

It will also

illustrate

some of the general principles which govern the application of anti-metabolites to the practical problems of chemotherapy.

28

In 1940, D. D. Woods [l] proposed the classical anti-metabolite theory that certain substances which prevent

cell-growth

(anti-metabolites) may do so by interfering with the formation of other substances essential for cell growth Inhibition occurs if the anti-metabolite

(metabolites).

structurally

resembles the metabolite and thus fits the same enzyme, and yet is sufficiently different to inhibit enzyme activity. Sulphanilamide, derived from prontosil in vivo. was the first anti-metabolite to be of clinical utility, and is, of course, related structurally to p-aminobenzoic

Prontosil

— >

acid.

M - ^ ^ - S O a N R j

Sulphanilamide

H a N - ^ ^ - C Q j H

p-Aminobenzoic

acid

Antimetabolite theory, however, has not led to many useful drugs over the past 30 years, and it is not always that anti-metabolites per se are necessarily agents.

recognized

chemotherapeutic

The success of the sulphonamides as anti-bacterial

agents is due to interference with a biochemical

reaction

which is present in the parasite but absent from the host. The basis of the rational approach to chemotherapy which is to be discussed in this paper is the exploitation of differences in the biochemistry of the parasite from that of the host.

Ideally, we are seeking a situation whereby an

enzymic system is present in the parasite but absent from the host and which may thus be specifically inhibited.

When

this ideal situation is not obtainable it is still possible to obtain substantial quantitative differences in the binding of inhibitor to enzyme when the enzyme is common to parasite and host.

29 THE

BIOSYNTHESIS

OF

TETRAHYDROFOLATE

COENZYMES

Coenzymes based on tetrahydrofolate are essential in the cell metabolism of both pathogenic micro-organisms and mammalian hosts.

their

They are involved in the transfer of one

carbon atom units in vivo and are 'thus vital for the biosynthesis of purine and pyrimidine nucleotides and for the biosynthesis of nucleic acids.

A

therefore

significant

difference, however, is the fact that many of these microorganisms synthesise dihydrofolate,

the precursor of tetra-

hydrof olate coenzymes, from simple precursors whereas

the

mammalian host is dependent on exogenous sources of folate [ 2 j .

The differences in the biosynthetic pathways of host and parasite are now well defined and are set out below.

Scheme

1 illustrates the route to tetrahydrofolate coenzymes found in most mammalian species.

The enzyme

dihydrofolate

reductase has proved to be of especial significance chemotherapy and will be discussed

in

later.

CO2H I

•CH

Diet

I

C

H,

CH-, co2H

Diet

Dihydrofolate

Reductase

H Diet

Coenzymes Scheme 1

30 Micro-organisms, on the other hand, synthesise dihydrofolate from simple precursors and the biochemistry of the final stages has been clarified considerably by the elegant work of G. M. Brown [3] and others [4] and is summarised in Scheme 2.

The enzymes of significance in this pathway are

dihydroneopterin aldolase,

hydroxymethyldihydropteridine

pyrophosphokinase, dihydropteroate synthase and dihydrofolate synthase.

One of these, the pyrophosphokinase will be

discussed in detail later.

Guanosine

triphosphate

O

(Ch0H)/Ha0H

w

Nk/CHzOH

HNT H

Aldolase

H pyrophosphokinase

0 UN/' lyj

"

A"

'j' -H

H

dihydrofolate

^

dihydropteroate xW./,A. , svntha.se M .tJ ^ N synthase H

X

synthase

-^-Coenzymes CI H

ch2

cO3H Scheme 2

31 The two biosynthetic pathways clearly have some steps which are common (from dihydrofolate onwards) and some which are distinctive or peculiar to the micro-organism.

Thus we

have here the required difference between parasite and host which can be exploited for chemotherapeutic purposes. must be noted that most pathogenic micro-organisms

It

cannot

use preformed folic acid because folic acid can only enter a cell by an active transport mechanism which is not present in most bacterial cells.

This fact is of great

if inhibitors of the enzymes in the biosynthetic are to be used as antibacterial agents.

importance pathway

The differences

in

the biosynthetic pathways are summarised in Scheme 3. Host

Parasite

Folic Acid

Simple pteridine

precursor

V 7,8-dihydropteroate >• 7, 8-dihydrof olate

-i

5,6,7,8-tetrahydrofolate Y Co-enzymes

ENZYME

INHIBITION

AND

CHEMOTHERAPY

Inhibitors of dihydrofolate reductase are of two types. Aminopterin and amethopterin are full structural analogues of the substrate whereas 2,4-diaminopyrimidine such as trimethoprim are not so closely related

derivatives structurally.

Both types are powerful inhibitors and give 50fo inhibition _9

of enzyme activity at about 1 x 10

M.

Their chemo-

therapeutic activities, however, are completely different.

32

CH,

R=H R=CH 3

CO,H

Amethopterin

Inhibitors of the aminopterin type possess a full complement of binding sites.

The binding of inhibitor to enzyme would

thus be expected to parallel that of substrate to enzyme whatever the source of the latter.

In fact, these

have chemotherapeutic utility only as anti-leukaemia

compounds drugs

in mammals, and show little toxicity to bacteria and protozoa. It has been shown that these molecules are transported

across

cell membranes by the same mechanisms which govern transport of folic acid.

Thus they affect only those species which

require exogenous folic acid, and have no effect on bacteria etc. which lack the active transport G. H. Hitchings and his colleagues

mechanism.

[5] demonstrated some 25

years ago that essentially all derivatives of the 2, 4-diaminopyrimidine ring system inhibit dihydrofolate reductase and moreover that the inhibition can be made highly

specific

towards the enzyme from one organism or another by relatively minor changes in structure.

This selectivity is no doubt

related to the fact that these inhibitors do not possess a full complement of binding sites. Inhibitors of this type are able to pass through cell walls by a passive diffusion process, i.e. progress into the cell is quite unrelated to the ability of the cell to assimilate preformed folic acid.

The origin of this selectivity has

been the subject of detailed study by J. J. Burchall

[6] and

appears to be due to differences, possibly even of primary structure, in the enzyme dihydrofolate reductase in the

33 organism in question. Ml-k Generalized

X

Structure

Examples of Inhibitors

il - O Me 'ONe

M

HjW^f^

Et

OMe Trimethoprim

Daraprim

INHIBITION OF DIHYDROFOLATE BIOSYNTHESIS In principle, a specific inhibitor of any one of the four enzymes involved in the conversion of dihydroneopterin into dihydrofolate should prove to be a selectively toxic agent and affect only the parasite. Sulphonamides are known to be effective inhibitors of dihydropteroate synthase [7].

Some aspects of our work with this

enzyme will be discussed in a paper [8] to be presented in this symposium.

later

The present study is concerned with

inhibitors of the pyrophosphokinase.

Prior to our own

investigations in this area, no attempts have been made to design specific inhibitors of this enzyme. The kinase catalyses the following reaction in which two phosphate residues are transferred to the hydroxymethylpteridine from adenosine

triphosphate.

34

K

2+

o

lh NI' X H

AMP

ATf

CH20(g)(£)

N

H

We have synthesised a number of structural analogues of the substrate for evaluation as potential inhibitors.

The

enzymes involved in the biosynthesis of 7,8-dihydrofolate appear to have an absolute requirement for binding of such analogues for the 2-aminopyrimidin-4-one moiety.

They are

thus quite distinct from dihydrofolate reductase where diaminopyrimidine derivatives bind

THE

CHEMISTRY

OF

THE

(a) Pyrimido[5.4-e]as

2,4-

strongly.

INHIBITORS

Triazines

The compounds all conformed to the following

generalized

structure.

•AY R'

Two synthetic methods were used although these differed in minor detail.

only

Thus, condensation of a 4-chloro—5-nitro-

pyrimidine with a monoacyl hydrazine gave the acyl hydrazinopyrimidine.

corresponding

This cyclised readily on

reduction of the nitro group to give the

dihydropyrimido-

35 triazine.

Oxidation to the fully aromatic compounds proved

to be difficult

However, oxidation of the 3-methyl deriv-

ative ( R = C H 3 ) was accomplished by bubbling air through a suspension of the dihydro derivative in ethanolic sodium hydroxide.

The aromatic compound was isolated as its

sodium salt.

o H M

N

A

^ N

0

• N 0,

+

HZN

NH

CO

R

^ ^

H I

NH

CI

NH CD

R

R=CH, CH2.C6 H 5

H N T

The second method led more conveniently to the dihydro derivatives.

V

N

Y

R

blocked'

This involved condensation of the

chloropyrimidine with 1,2-dimethylhydrazine to give a hydrazinopyrimidine.

Acylation,followed by reduction and

cyclisation,gave the required compounds. The precise structure of the dihydropyrimidotriazines has given us some cause for concern.

In each case bar one, the

ultraviolet absorption spectra, typicallyJ X

279 nm at max pHl and X 280 nm at pH13, are anomalous with maxima at max ' shorter wavelengths than would be expected for the structures shown above.

This fact, together with the observation that

these molecules tenatiously retain one molecule of water,

36

M e N H MI1

Me

-H N" fN t n*— N / H 2

V

R=CH-, CH2,C6 H 5 HNJ'

rie

H,N

N

suggests that dehydration has not followed cyclisation as is usual in such heterocyclic systems.

It seems likely, there-

fore that we have hydrated structures such as that shown below. o

H

itc °

H N *

^

N

M

e

I I HO-C N (Me i R It is unusual in pteridines to find stable hydrated

species

with a substituent R in the position shown and we have therefore considered the isomeric structure shown on the right. At the present time there appears to be little evidence in favour of such structures.

The

dihydropyrimidotriazines,

which are insoluble in water, dissolve readily in alkali .indicating the presence of an acidic hydrogen atom, one of the dihydro derivatives can be oxidised (see above) to a fully aromatic compound isolable as a sodium salt, and one

37 of the dihydro derivatives (R-CH 2 .0H) prepared in exactly the same way, does not retain a molecule of water and shows a normal ultraviolet absorption spectrum Xmax 310, ' 281 and A 7 233 (pHl) and Xma -v- 301, 281 and 227 nm (pH13).

(to) Blocked Dihydropteridines The compounds all conformed to the following generalized struc ture.

o A

m

H ^ r A i hN ^ The synthesis of this type of blocked dihydropteridine is conveniently achieved by a modification of the method described by Boon et al [9] for the preparation of 7,8dihydropteridines.

This method, which has also been used

by Pfleiderer and Zondler [10] for the synthesis of a blocked dihydropteridine, involves cyclisation of an appropriately substituted pyrimidine which is itself prepared by condensation of an oc-aminoketone with a pyrimidine bearing an activated chloro group.

CO

Y N ^ C I

R

R

(N)

N R

k

i

N'

CO -R

NIT

x >k

Y ^ n

•NH

R

38 The required cc-aminoketones are not readily available and we have investigated several routes to compounds of this type. (a) The first of these involved preparation of 2-methyl-2phthalimidopropanoic acid and its conversion into 1-hydroxy3-methyl-3-phthalimidobutan-2-one

by successive treatment with

thionyl chloride, diazomethane and dilute hydrochloric

acid.

Stronger acid hydrolysis removed the protecting group and gave the required a-aminoketone which was converted into its semicarbazone to prevent self-condensation

reactions.

Reaction with 4-chloro-5-nitropyrimidine followed by hydrolysis of the semicarbazone, reduction of the 5-nitro group and cyclisation gave the required pteridine derivative

o f b - c - c o ^

b

H

ch,

CH,

NJ

/

C H,

N - C - C O . ^ O H O

CHs N M H C O . N H , I II ® H 3 N - C — C — C H . D H

at 100

X

10

M

c h

3

5o/

at

U

X

10

M

3

50io at

15

X

10

M

CH2 • CH3

50 io at

2

X

10

M

CH2•CH^

50$ at

5

X

10

M

37% at 100

X

10

M

c h

spirocyclohexyl

0

We thus see that we have here a series of very effective enzyme inhibitors, the best of which give 50$> inhibition of enzyme activity at concentrations of 2-5/O-M.

Further studies

are in progress in an attempt to find still more

effective

inhibitors.

Activity against whole

Organisms

It is disappointing to report that the biological activity of the blocked dihydropteridines against a micro-organism as S. Aureus is very low indeed.

What is very

such

significant,

however, is the synergistic effect which is observed when these compounds are used in combination with other known antibacterial agents such as trimethoprim which

inhibits

dihydrofolate reductase and sulphamethoxazole which dihydropteroate synthase.

Some preliminary results are

shown in the following table. experiment was

The pteridine used in this

2-amino-4-hydroxy-6-hydroxymethyl-7,7-dimethyl-

7,8-dihydropteridine

(DMHP).

DrugyfciM

[1/HjPtCHjOPP] /iM"'

^

intercepts

from plot (B) at the various H2PtCH2 0 P P levels, and vice-versa. The standard errors of the slopes ranged from 3-17% (mean = 7.5%) and the slopes within each plot did not differ significantly (p = 0.05). However, one experiment is seemingly inconsistent with the ordered reaction sequence; the initial velocity plots of each substrate at several fixed levels of the alternate substrate (Fig. 6).

A sequential mechanism

normally gives converging lines in double reciprocal plots of this type (17), yet these data appear to yield parallel lines, which is usually indicative of a ping pong mechanism in which the first product is released before the second substrate binds to the enzyme. may be offered for this apparent inconsistency.

Several explanations These data cannot dif-

ferentiate between parallel lines and lines in which the slopes differ only slightly.

Henderson et at. (18) argued that unless the ratio of the

true dissociation constant to substrate concentration is >0.1, then any deviation from parallel lines is extremely difficult to detect.

Thus if

the dissociation constants for H 2 ptCH 2 0PP and for pAB are less than about 0.05 pM, convergent lines would not be observed.

A proposal of alternate

reaction sequence was offered to explain parallel line initial velocity plots with human hypoxanthine phosphoribosyltransferase

(19).

Cleland

(17) has discussed the conditions under which parallel lines are observed in initial velocity plots.

In the absence of other evidence {e.g. from

isotope exchange and equilibrium dialysis studies) the mechanism of reaction of H2~pteroate synthetase cannot be considered firmly established, although the product inhibition studies indicate an ordered reaction. I | The role of Mg

in this reaction has been in some doubt since an abso-

lute requirement has been claimed for the E. aoli enzyme (5), but not for

68 Fig. 7. Effects of divalent cations on the activity of E. oo~li dihydropteroate synthetase. The salts indicated were included in the standard enzyme assay systems (30 min. incubation) at 25, 200, and 2,500 yM, and the amount of dihydropteroate synthesized compared to a control with no salts added.

25

250

2500

C0NC, ;u.M

partially purified preparations of this enzyme from other sources (2,3,4). Until now we could only observe a 15-30% stimulation by MgCl2 of the activity of H2~pteroate synthetase from E. aoli,

even with enzyme prepara-

tions which had been further purified by DEAE-cellulose chromatography (5) and/or extensively dialyzed.

However, recently a number of experiments

have been performed in which the activity observed in the absence of MgCl2 was only 1/10 - 1/3 of that obtained with MgCl2 present. The activity in I | the absence of added Mg is inhibited by low levels of EDTA (0.5 - 20 yM) I [ or 1 mM ATP and this inhibition can be reversed by the inclusion of Mg in the reaction mixtures. of Mg

Maximal rates can be obtained with low levels

; Fig. 7 demonstrates full activity with 25 yM MgCl2 or MgSO^.

Other experiments have demonstrated detectable stimulation of activity at | | I j levels as low as 1 yM.

Mn

can substitute for Mg

, but the other diva-

lent cations tested only had small effects or were inhibitory (Fig. 7). I | Thus Mg

does seem to be required for the H2-pteroate synthetase from

E. aoli.

The variable results I | obtained may have been due to the intro-

duction of low levels of Mg

in incubation mixtures as a contaminant of

other components added. SUMMARY Dihydropteroate synthetase activity of E, aoVi

and P. bevghei

bited by H2~pteroate, H2-folate and H2~homopteroate.

was inhi-

Dihydrohomopteroate

was a strong inhibitor of the synthetases from both sources, competitive with H2PtCH20PP, and apparent K^ values of 0.15 yM and 7 nM were found for the enzymes from E. coli,

and P. bevghei,

respectively.

The activity of

69 H2~pteroate synthetase was inhibited by its products in a pattern consistent with an ordered mechanism.

The assignment of H2ptCH20PP as the first

substrate to bind to the enzyme and H2-pteroate as the last product to leave, was supported by the finding that both of these compounds protected the enzyme from heat denaturation, but pAB and Na pyrophosphate did not protect.

However, double reciprocal plots of each substrate at several

fixed levels of the other substrate gave lines in which the slopes were not significantly different, suggesting a ping pong mechanism.

Therefore,

other evidence is required to determine the reaction mechanism of this enzyme. Studies with divalent cations and chelating agents indicate a requirement | |

for low concentrations of Mg

or Mn

, which might have been present in

previous studies as contaminents of some other components of the reaction sys tems. REFERENCES 1.

Brown, G.M.:

The Biosynthesis of Pteridines.

Advances in Enzymology

35, 35-77 (1971). 2.

Shiota, T., Baugh, C.M. , Jackson, R., and Dillard, R.:

The Enzymatic

Synthesis of Hydroxymethyldihydropteridine Pyrophosphate and Dihydrofolate. 3.

Biochemistry J3, 5022-5028 (1969).

Ortiz, P.J. : Dihydrofolate and Dihydropteroate Synthesis by Partially Purified Enzymes from Wild-type and Sulfonamide-resistant Pneumococcus. Biochemistry 9, 355-361 (1970).

4. McCullough, J.L., and Maren, T.H.: Plasmodium bergheii and Sulfadiazine. 5.

Dihydropteroate Synthetase from

Isolation, Properties and Inhibition by Dapsone Mol. Pharmacol. _10, 140-145 (1974).

Richey, D.P., and Brown, G.M.:

The Biosynthesis of Folic Acid. IX.

Purification and Properties of the Enzymes Required for the Formation of Dihydropteroic Acid. 6.

J. Biol. Chem. 244, 1582-1592 (1969).

Friedkin, M., Crawford, E.J., and Plante, L.T.: Approaches in Cancer Chemotherapy.

Emperical vs.

Rational

Ann. N. Y. Acad. Sci. 186, 209-

213 (1971). 7. Kisliuk, R.L., Friedkin, M., Schmidt, L.H., and Rossan, R.N.: ' il Activity of Tetrahydropteroic Acid.

Anti-

Science 156, 1616-1617

70 8.

Ferone, R.:

The Enzymic Synthesis of Dihydropteroate and Dihydro-

f o l a t e by Plasmodium berghei. 9.

Cleland, W.W.: Data.

10.

J. Protozol. 20, 459-464 (1973).

Computer Programmes f o r Processing Enzyme Kinetic

Nature 298» 463-465 (1963).

Nahas, A., and Friedkin, M.:

Fate of Tetrahydrohomofolate in Mice.

Cancer Res. 29, 1937-1943 (1969). 11.

Zakrzewski, S.F., and Sansone, A.M.:

A New Preparation of Tetra-

hydrofolic Acid (Volume 18 of Methods in Enzymology, ed. by McCormick, D.B., and Weight, L.D.). 12.

Part B, 728-731 (1971).

Shiota, T . , Disrealy, M.N., and McCann, M.P.:

The Enzymic Synthesis

of Folate-like Compounds from Hydroxymethyldihydropteridine Pyrophosphate. 13.

J. Biol. Chem. 239, 2259-2266 (1964).

Suzuki, Y . , and Brown, G.M.:

The Biosynthesis of Folic Acid X I I .

Purification and Properties of Dihydroneopterin Triphosphate Pyrophosphohydrolase. 14.

J. Biol. Chem. 249, 2405-2410 (1974).

Mathis, J.B., and Brown, G.M.:

The Biosynthesis of Folic Acid XI.

Purification and Properties of Dihydroneopterin Aldolase.

J. B i o l .

Chem. 245, 3015-3025 (1970). 15.

Webb, S., and Ferone, R.:

16.

Nixon, J.C., Butz, R.F., Nichol, C.A., Dev, I . K . , and Harvey, R.J.: In vitro

Inhibition of E. eoli

Reduced Folates. 17.

Unpublished observations.

Cleland, W.W.:

GTP Cyclohydrolase by Several

Biophys. J. _15, 11a (1975). Steady State Kinetics in The Enzymes: Kinetics and

Mechanism (Boyer, P.D., ed.) Vol. 2, pp 1-61, Academic Press, N. Y. (1970). 18.

Henderson, J . F . , Brox, L.W. , Kelley, W.N., Rosenbloom, F.M., and Seegmiller, J.E.: ribosyltransferase.

19.

Kinetic Studies of Hypoxanthine-Guanine PhosphoJ. Biol. Chem. 243, 2514-2522 (1968).

Krenitsky, T.A., and Papaionnou, R.:

Human Hypoxanthine Phospho-

ribosyltransferase I I . Kinetics and Chemical Modification. Chem. 244, 1271-1277 (1969).

J. Biol.

71 DISCUSSION *

Poe: Dr. Ferone deserves to be congratulated for this beautiful kinetic data on a most difficult assay. Have you detected another product of the enzymatic reaction ? If there were another product, it could explain the discrepancy between your initial rate studies (indicating "ping-pong" mechanism) and your product inhibition studies. Ferone: We have no evidence for this and I am not sure that this would lead to parallel lines in the initial velocity studies. Mautner: Can this reaction be run backwards ? Then one could use labelled pyrophosphate to determine whether, in absence of PABA, pyrophosphoric pteridine is formed ? Ferone: We have not tried this, but others have published that the reaction is not reversible. However, such an experiment can also be run in the forward direction, and we hope to do so. Mautner: Have you looked at esters of pteroic or homopteroic acid as inhibitors of your enzyme ? Ferone: No, we have not. Degraw: Have you any explanation for activity of homopteroates against malaria organisms ? Are the compounds penetrating the cell wall ? Ferone: The enzyme inhibition data certainly suggests that fy-homopteroate inhibits by interference with folate biosynthesis. By implication then one would suppose the compound penetrated the cell membrane, but of course this is no proof.

Dihydropteroate Synthase: Purification by Affinity Chromatography and Mechanism of Action C.J. Suckling, J.R. Sweeney and H.C.S. Wood

Dihydropteroate

synthase (E.C. 2.5.1.15) is an enzyme of

especial significance from the point of view of chemotherapy, because it has been shown to be strongly inhibited by sulphonamides, and is the principal site of action of these antibacterial agents

[1].

This enzyme is one of those involved in the biosynthesis tetrahydrofolate

of

in bacteria and other parasites (Fig. 1).

The use of specific inhibitors of one or more of these enzymes in a new approach to chemotherapy has been discussed earlier in this symposium

[2].

It is clear that the design

of such inhibitors will be greatly facilitated by a detailed understanding of the structure and properties of the enzyme in question.

Thus there has been enormous

interest

in the enzyme dihydrofolate reductase which is specifically inhibited by the successful antibacterial agent [3].

trimethoprim

The primary structure of the enzyme from E.Coli has

now been established

[4], and crystallographic

studies to

elucidate the conformation of the enzyme are underway. We plan a similar investigation of dihydropteroate

synthase

and this paper is concerned with the purification of the enzyme and some studies of its mechanism of action.

74

HN

N.

Y

H' A N A

^ NX'

CH,OH

/\

n

H

Kinase

ATP

CH 2 OPP

HN

__

I

* 7 AMP "2

>-C02H

H (DHPP)

(PAB) Dihydropteroate Synthase

Sulphonamides Inhibit

N

Hi/"

A

H, r r ^ N ^

N

H D ihydro folate Synthase

Dihydrofolate Reductase Tetrahydro- < folate

0

I

NO. / C H , NH

V"Y

Trimethoprim h ij N ^ ^ ^ N Inhibits H

N/

2

-CONH.CH

H H

CH,

CHgCOjH

Figure 1 AFFINITY

CHROMATOGRAPHY

Although dihydropteroate synthase has been purified by classical enzymological techniques [5], it is difficult to separate it from the preceding enzyme in the biosynthetic pathway, the pyrophosphokinase.

We have

therefore

investigated its purification by the biospecific of affinity chromatography

technique

[6] using a sulphonamide

inhibitor

attached to Sepharose. Unlike conventional techniques for purifying enzymes which rely upon physico-chemical differences such as charge and size between proteins, affinity chromatography makes use of the ability of enzymes to bind their substrates strongly at the active site.

Further, enzymes are specific with regard

to the substrate that they will accept. enzyme-substrate

This

specific

interaction forms the basis of enzyme

75 purification by affinity chromatography.

Fortunately, not

only substrates but also inhibitors can bind;

the inhibitors

may be substrate analogues or transition-state analogues

[7].

In our work we have used a substrate analogue. In order to carry out affinity chromatography, the substrate or inhibitor (the ligand) is covalently attached to an inert polymer matrix in such a way that binding of the ligand to the enzyme is not impaired.

A

'spacer-arm'

is

usually employed to extend the ligand away from the polymer backbone.

This material is packed into a column and the

crude enzyme preparation is applied (Fig. 2 A ) .

AS the

proteins pass down the column the enzyme is specifically retained by binding to the ligand (Fig. 2B) , the unwanted protein being eluted.

When all of this protein has been

removed, a column of enzyme bound through the ligand to the polymer matrix remains.

The enzyme can be eluted by

changing the buffer so that the enzyme no longer binds to the ligand (Fig. 2c).

W \

AT

A/

AT

(A)

(B) Figure 2

(C)

76 Our purification of dihydropteroate synthase uses the sulphonamide

(l) as ligand.

with cyanogen bromide

Sepharose 4B was activated

[8] and coupled to the spacer-arm

di(3-aminopropyl)amine

[9].

The sulphonamide was condensed

with the Sepharose conjugate using

l-cyclohexyl-3 - (2-morphol-

inoethyl) carbodi-imide metho-jD-toluenesulphonate aqueous dioxan.

in 50$>

The product thus obtained contained

1.2-

1.7//.M of inhibitor per ml. of settled gel.

-OH

H 2 N ( C H 2 ) 3 NH ( CH 2 ) 3 N H 2 2=NH

— 0

-O.CONH(CH2 ) 3 N H ( C H 2

)3NH2

(l) + Carbodi-imide -OH —

O.CONH(CH2 ) 3 N H ( C H 2

NHSO,

)3NHCO.CH2NHSO.

NH,

Figure 3 The sulphonamide (K±

8 x 10

M).

(l) is a potent inhibitor of the enzyme Despite this, when a saturated solution of

crude enzyme from E.Coli in either 0.2M Tris-HCl buffer, pH 8 . 5 ,

or 0.1M potassium phosphate buffer, pH 8 . 0 ,

applied to a 60 x 8 mm. column of the

was

sulphonamide-Sepharose,

the protein and enzymic activity were eluted

together,although

the enzyme was slightly retarded by the column (Fig. 4 A ) . Protein was determined by the method of Lowry [ 1 0 ]

and enzyme

77 activity was assayed using

1k

C labelled jD-aminobenzoic

following the method of Richey and Brown [5].

acid

However, when

the buffer contained in addition 18yulM 2-amino-4-hydroxy-6hydroxymethyl-7,8-dihydropteridine 5mM dithiothreitol

pyrophosphate

(DHPP) and

(DTT) to prevent oxidation of the dihydro-

pteridine, essentially all the enzyme activity was retained on the column whilst the bulk of the protein was eluted. Immediately upon removal of DHPP and DTT from the buffer, enzymic activity emerged from the column (Fig. 4B).

This is

good evidence for a biospecific process and represents a purification of the enzyme of 180 fold in one step.

TRANSMITTANCE

(#)

280 run.

- 90

•in——n

ENZYME

~iSL L

"35

ACTIVITY (tip. m.) -18 .«M DHPP * 5 nM DTT

-

TRANSMITTANCE AT 280 nm. INCLUDFS PROTEIN * DHPP ABSORPTIONS

RI!

2

Figure 4

25 30 Column Volumes

1)0

78 If the substrate DHPP can induce binding of the inhibitor to the enzyme, it was obviously of interest to see whether analogues of DHPP and related compounds would do likewise. Of the compounds which we have tested only the monophosphate analogue of DHPP gives rise to binding of enzyme to sulphonamide inhibitor, and hence allows affinity (Fig.

chromatography

5).

Binding of enzyme

Compound

Concentration used in affinity chromatography

0

11

CH 2 OP

5.9 x 10

Yes*

H PPi

A H

2

.N^

_3

No

No

M

1 x 10

M

pteridine 1.2x10 pyrophosphate 2.4x10

Me

_5

No

k x 10

No

8 x 10

M

/ ^ N / ^ N ^ M e H nh2

_5

M

OMe *

Figure 5

I 4 0 = 8 x 10

-5

M

_3

3M

M

79 With this information to hand, we wished to find out whether a column prepared using p-aminobenzoic acid, one of the substrates of the enzyme, as ligand could give rise to affinity chromatography.

However, a column of p-amino-

benzoic acid - Sepharose, prepared as in Figure 3, failed to achieve affinity chromatography under identical conditions to those successful with the sulphonamide - Sepharose column. This is not surprising since amides derived from p-aminobenzoic acid are known to have little antibacterial activity [11]. Finally, it was important to check whether the linking arm, di-(3-aminopropyl)amine, was contributing to the binding of the enzyme.

We were surprised to find that a column of

aminoalkyl-Sepharose completely retained all protein and all enzymic activity.

Both were eluted with 1M sodium chloride.

However, this column did not retain enzyme activity when the buffer contained DHPP and DTT although significant occurred.

trailing

This result suggests that some non-specific

binding is taking place.

MECHANISM

OF

ACTION

The obligatory presence of DHPP in the enzyme solution applied to the affinity column and the negative elution effected by its removal suggest that an ordered kinetic mechanism (Fig. 6) may be operating in which the pteridine pyrophosphate DHPP must bind to the enzyme before p-aminobenzoic acid or sulphonamide. DHPP

PAB

i

Dihydropteroate + PP.

i

FDHPP

„DHPP PAB Figure 6

'

PP Pteroate

80 Similar chromatographic behaviour has been observed with lactate dehydrogenase experiments

[12].

In this case, kinetic

[13] confirm that NADH must be bound to lactate

dehydrogenase before pyruvate binds and accordingly pig heart lactate dehydrogenase only binds to an inhibitor (oxamate) containing column in the presence of NADH. Our results would appear to have an important bearing on the mode of action of the anti-bacterial sulphonamides and we are currently investigating this aspect of the problem. are also developing the affinity chromatography for large scale purification of dihydropteroate synthase and hope to obtain sufficient enzyme for affinity labelling and crystallographic

studies.

Acknowledgement We thank the Science Research Council for the award of a studentship (to JRS) and Dr. R. Ferone of the Wellcome Research Laboratories, North Carolina for crude E.Coli preparations.

REFERENCES 1. Brown, G. M:

The biosynthesis of folic acid. II.

Inhibition by sulphonamides.

J. Biol. Chem.,

237.

536-540 (1962). 2. Wood, H. C. S: biosynthesis.

Specific inhibition of dihydrofolate A new approach to chemotherapy.

This Symposium, pp.

We

81

3. Burchall, J. J. and Hitchings,G. H:

Inhibitor binding

analysis of dihydrofolate reductases from various species.

Mol. Pharmacol., _1, 126-136

Bennet, C. D:

(1965).

Similarity in the sequence of E. Coli

dihydrofolate reductase with other pyridine nucleotide requiring enzymes.

Nature, 248. 67-68

5. Richey, D. P. and Brown, G. M: acid. IX.

(l974).

The biosynthesis of folic

Purification and properties of the enzymes

required for the formation of dihydropteroic J. Biol. Chem., 244. 1582-1592 6. Lowe, C. R. and Dean, P. D. G: Wiley-Interscience, New York,

acid.

(l969). 'Affinity Chromatography' 1974.

7. Wolfenden, R: Analog approaches to the structure of the transition state in enzymic reactions.

Accounts of Chem.

Research, ¿ , 10-17 (l972). 8. Cuatrecasas, P:

Protein purification by affinity

chromatography, J. Biol. Chem., 245• 3059-3065

(l970).

9. Steers, E., Cuatrecasas, P. and Pollard, H. D:

The

purification of (3-galactosidase from Escherichia by affinity chromatography.

coli

J. Biol. Chem., 246.

196-200

(1971). 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J:

Protein measurement with the Folin Phenol

J. Biol. Chem., 192, 265-275 11. Northey, E. H:

(l95l).

Structure and chemotherapeutic

of sulphanilamide derivatives. (1940).

reagent.

activities

Chem. Revs., 2J_, 85-197,

82 12. o'Carra, P. and Barry, S: lactate dehydrogenase.

Affinity chromatography

of

FEBS Letters, 21, 281-285

(1972). 13. Schwert, G. W. in 'Pyridine

Nucleotide-Dependent

Dehydrogenases', Ed. Sund H., Springer Verlag,

Berlin,

133 (l970). DISCUSSION Poe: What is the biological source ? Is this an "induced" strain with higher levels of enzyme ? Wood: The enzyme was obtained from E.coli. This was provided by Dr. R. Ferone and was not an induced strain. G.M.Brown: ^-pteroate synthase is a relatively unstable enzyme in our hands. My question is: how stable is your 200-fold purified enzyme ? Wood: No investigations have been made, as yet, on stability of the purified enzyme. The instability to which you refer may explain a purification factor (x 200) which is made for an efficient affinity column. G.M.Brown: A second question is: what was the recovery of enzyme activity from the affinity column ? Wood:

I regret that I do not have this information available.

Whiteley: Do you know the molecular weight ? Wood: Rickey and Brown have quoted a molecular weight of 5o.ooo for dihydropteroate synthase. Our material has not been examined as yet. Whiteley: In relation to this, have you altered the spacer length ? Wood: No variation has been made in the length of the spacer arm. Whiteley: Have you blocked xs spacer ion-exchange groups ? Wood: Excess spacer arm groups have not been blocked and presumably ionexchange effects are responsible for slight retardation shown in figure 4A. Shiota: Has the affinity chromatography purified dihydropteroate synthetase been tested with p-aminobenzoylglutamate ? Wood: No, it has not been so tested. Scrimgeour: There is a phenomenon called "substrate synergism" noted by Boyer and Bridger and apparently observed with several enzymes, in which

83 the association constants for substrates are increased when all substrates are present. Could this be an explanation of your binding only in the presence of DHPP, and might it also have some effect on Dr. Ferone's kinetic anomaly ? Wood: I believe that the "ordered kinetics" reported by Dr. Ferone, and the results of our affinity chromatography can be explained by assuming that the DHPP changes the conformation of the enzyme so that the sulfonamides (and presumably also the PAB) is "recognized". This would appear to be a form of "substrate synergism" where one substrate must bind before the other.

The Properties of Gamma Glutamyl Hydrolase (Conjugase) from Bovine Liver P.B. Rowe, M. Silink and R. Reddel

Several years ago our research group decided that the biologically significant and metabolically active forms of the mammalian folates were their polygammaglutamyl derivatives. The unique polygammaglutamyl peptides, which have been extensively studied by protein physical chemists (1) are polyanions. It has been shown that, in the range of sizes we are concerned with in folic acid metabolism, that (at the physiological pH range) these peptides assume an extended rod configuration maintained by the free alpha carboxyl groups provided the ionic strength of their environment is not too high (1). A major objective of our studies has been to define a biological role for these molecules. We have undertaken a systematic examination of pteroylpolyglutamates in the mammal from four different aspects: 1.

Mechanisms of biosynthesis.

2.

Mechanisms of degradation.

3.

The role of polyglutamates as the natural substrates for the folate interconversion enzymes.

4.

Other potential roles for the glutamyl peptides, e.g. as modulators of the membrane transport system for folic acid derivatives.

The second area of study demanded the purification and characterisation of gamma glutamyl hydrolase (conjugase), the enzyme responsible for the cleavage of the gamma bonds. We selected the enzyme from liver as we considered that as this organ contained the bulk of the body folates then the enzyme involved in the cleavage of the glutamate residues should be central to any control mechanisms for regulating folate flux. Beef tissue was selected as our preliminary studies indicated a close similarity between this enzyme and the human enzyme examined by Baugh and Krumdieck (2), and our work is aimed at establishing an understanding of folate metabolism in Man. It must be acknowledged that none of this work would have been possible without the pioneering studies of Baugh and his colleagues who established the methodology for the solid phase synthesis of pteroylpolyglutamates.

86 P u r i f i c a t i o n o f t h e enzyme was a c h i e v e d by t r a d i t i o n a l methods o f p r o t e i n p u r i f i c a t i o n whose u t i l i t y was e x t e n d e d by t h e r e v e r s i b l e m o d i f i c a t i o n o f t h e enzyme w i t h t h e p o l y a n i o n i n h i b i t o r , Blue Dextran. The f u n c t i o n a l g r o u p s o f t h i s m a t e r i a l a r e t h e s u l f o n y l g r o u p s on t h e a n t h r o q u i n o n e d y e , C i b a c r o n B l u e 3GA. The B l u e D e x t r a n can be r e a d i l y removed f r o m t h e enzyme by t h e a d d i t i o n o f b o v i n e serum albumin t o t h e assay system. The d e t a i l s o f t h e p u r i f i c a t i o n t e c h n i q u e and r a n g e o f a f f i n i t y chromatography methods we u s e d a r e c u r r e n t l y i n p r e s s ( 3 ) and w i l l not be d i s c u s s e d h e r e . The a s s a y system employed was a m o d i f i c a t i o n o f t h a t d e s c r i b e d by Baugh and h i s c o l l e a g u e s ( F i g . 1 ) .

GittiA GLUTAMYL UARBOXYPEPTIHASE AS3iI 1.

Pte - GluO- GluO- UC14- Glu - ^Glu

2.

Pte - (Glu)n + U014- Glu + Glu

3.

Fig.

(charcoal - Pte - (Glu)n)

1.

Principle

of

+

enzyme

+ Bnzyae +

T.C.A. Charcoal

UC14- Glu

assay.

The p r i n c i p l e i s t h a t ^ C - g l u t a m i c a c i d i s r e l e a s e d f r o m t h e s u b s t r a t e , t h e uncleaved p t e r o y l g l u t a m a t e s are adsorbed onto c h a r c o a l , removed by f i l t r a t i o n and t h e r e s i d u a l r a d i o a c t i v i t y measured. There i s a d e f e c t i n t h i s assay system i n t h a t t h e b o v i n e h e p a t i c enzyme i s a l s o an e n d o p e p t i d a s e and t h e r e f o r e p o l y g l u t a m a t e s a r e r e l e a s e d f r o m t h e pteroylpolyglutamate substrate. We e s t a b l i s h e d t h a t 25 p e r c e n t o f d i g l u t a m a t e , 70 p e r c e n t o f t r i g l u t a m a i e and 90 p e r c e n t o f t e t r a g l u t a m a t e would be a d s o r b e d t o t h e c h a r c o a l o v e r t h e c o n c e n t r a t i o n r a n g e s used i n t h e a s s a y s . A c c o r d i n g l y t h e r e l e a s e o f 14-C-glutamic a c i d i s not a t r u e r e f l e c t i o n o f t h e r a t e o f gamma bond c l e a v a g e . T h i s was t o some e x t e n t overcome by e x a m i n i n g t h e p r o d u c t s o f t h e r e a c t i o n by b o t h h i g h v o l t a g e e l e c t r o p h o r e s i s and t h i n l a y e r chromatography i n a number o f s y s t e m s . We a l s o u t i l i s e d a wide v a r i e t y o f d e r i v a t i v e s o f d i f f e r e n t glutamyl chain l e n g t h containing t h e r a d i o l a b e l l e d glutamic acid in a v a r i e t y o f p o s i t i o n s and s u b s t r a t e s c o n t a i n i n g a l p h a as w e l l as gamma b o n d s . We have e s t a b l i s h e d t h e f o l l o w i n g p r o p e r t i e s o f t h e

enzyme:

1. The enzyme i s l o c a t e d p r i m a r i l y i f not e n t i r e l y i n t h e lysosomes. T h i s has been e s t a b l i s h e d c o n c l u s i v e l y f o r r a t l i v e r u t i l i s i n g T r i t o n WR1339 t o a l t e r t h e d e n s i t y o f t h e s e o r g a n e l l e s ( 4 ) and by n e c e s s a r i l y c r u d e r f r a c t i o n a t i o n

87 studies with beef liver. In keeping with this intracellular localisation the pH optimum is acid at pH 3»9 (in 33 mM sodium acetate buffer) with a rapid fall in activity above pH 4.5« It must be noted that the pH optimum shifts to 4.5 in the inhibitory buffer sodium citrate. The protein is highly thermostable losing only 30 percent of its activity over 30 minutes at 6 5 ° . The "apparent" temperature optimum was 70°. This property is evidence of the primitive evolutionary origin of the enzyme consistent with its wide phylogenetic distribution, and is reminiscent of the properties of the thermophilic bacterial proteases which are usually active at the neutral pH range. The Km for Pte-Glu-Glu-Glu was 1.0 pM with a turnover number of 750 gamma bonds cleaved per min per molecule of enzyme. There is extreme anion sensitivity, a property probably related to the polyanionic nature of the substrate. 2. The enzyme contains reactive sulfhydryl groups which are related to the active site. Heavy metals are strongly inhibitory and p-CMB (1.0 x 10~4 M ) produces instant and total inhibition. p-CMB inhibited enzyme is stable and activity can be restored instantly by the addition of thiols for periods of at least 12 months. Thiols, such as ^-mercaptoethanol are essential for enzyme stability. 3. The enzyme is a glycoprotein, another property consistent with its lysosomal localisation. The protein band on polyacrylamide gel electrophoresis is PAS positive. There is a 1:1 stoichiometry of binding to Concanavalin A which was demonstrated by gel chromatography when the enzyme-Con A complex eluted at a MW of 160,000 which corresponded to one molecule of enzyme (MW 108,000) and one molecule of Con A (MW 55,000). This chromatographic behaviour also confirmed that the carbohydrate binding groups were involved in the enzyme binding by Concanavalin A. 4. The molecular weight is approximately 108,000. This figure is based on gel filtration experiments on Sephadex and polyacrylamide and does not allow for any effect produced by the carbohydrate residues on the enzyme. 5. Z n + + stabilises the enzyme. Detailed dialysis experiments against 1,10-o-phenanthroline demonstrated that the inhibition produced by long term exposure to this chelator could be reversed by Zn++. Ni++, C a + + and Mg++ ions but not CO++ could also partly restore the activity.

3e

Days

F i g . 2. E f f e c t on enzyme a c t i v i t y of l o n g t e r m d i a l y s i s against the chelator 1,10-o-phenanthroline and a t t e m p t e d r e a c t i v a t i o n by s p e c i f i c d i v a l e n t cations. • • enzyme a c t i v i t y a s a f u n c t i o n of t i m e of d i a l y s i s a g a i n s t o - p h e n a n t h r o l i n e e x p r e s s e d a s a p e r c e n t a g e of c o n t r o l a c t i v i t y . • • , o o c o n t r o l s o l u t i o n ( 1 . 0 mM z i n c c h l o r i d e , 5 . 0 mM p - m e r c a p t o e t h a n o l ) • 1 . 0 mM n i c k e l c h l o r i d e ; • • 1 . 0 mM c a l c i u m chloride; • • 1 . 0 mM c o b a l t c h l o r i d e . All s o l u t i o n s c o n t a i n e d 5 . 0 mM ^3-me r c a p t o e t h ano 1 . Z i n c c h l o r i d e 1 . 0 mM, was r e q u i r e d t o p r e s e r v e enzyme a c t i v i ty. A t o m i c a b s o r p t i o n s t u d i e s on t h e p u r i f i e d enzyme s u g g e s t e d t h e p r e s e n c e o f four' ( 4 ) z i n c a t o m s p e r m o l e c u l e o f MW 1 0 b , 0 0 0 . 6.

Enzyme-substrate

interaction:

(a) The enzyme a c t s s p e c i f i c a l l y on gamma b o n d s . Experiments with Pte-Glu-Glu-Glu c o n t a i n i n g a l p h a bonds in e i t h e r p r o x i m a l or d i s t a l p o s i t i o n s i n d i c a t e d t h a t t h e r e was l i m i t e d c l e a v a g e o f t h e t e r m i n a l gamma bond i n t h e p r e s e n c e o f a p r o x i m a l a l p h a bond w h i l e t h e r e was l i t t l e o r no c l e a v a g e o f t h e i n t e r n a l gamma bond i n t h e p r e s e n c e o f t h e t e r m i n a l a l p h a bond ( F i g . 3 ) .

STRUCTURE

C'feE.lEftt.E- &

F i g . 3Enzymatic c l e a v a g e of p t e r o y l t r i g l u t a m a t e s c o n t a i n i n g an a l p h a g l u t a m y l b o n d i n e i t h e r t h e p r o x i m a l ( Pte- C - G l u - G l u ) p o s i t i o n s when c o m p a r e d w i t h t h e n o r m a l gamma b o n d e d s u b s t r a t e (Pte-Glu-Glu-Glu). A consideration of the structural implications of t h i s l e d to conclusions similar to those reached f o r the mechanism o f a c t i o n o f C a r b o x y p e p t i d a s e A ( 5 ) .

data

(b) Longer p o l y g l u t a m y l chains are t h e p r e f e r r e d s u b s t r a t e T h i s study employed t h e s u b s t r a t e s P t e - G l u - l?-4cj-Glu-GluG l u - G l u and P t e - G l u - G l u - [ _ 1 4 C ] - G l u and e x p l o i t e d t h e d e f e c t i n t h e r o u t i n e assay in t h a t f r e e p o l y g l u t a m a t e s were p r e f e r e n t i a l l y a d s o r b e d t o t h e c h a r c o a l and h e n c e n o t d e t e c t e d in t h e assay system ( v i d e s u p r a ) . Accordingly the r e l e a s e o f r a d i o a c t i v i t y was d e l a y e d w i t h t h e l o n g e r c h a i n s u b s t r a t e c o n t a i n i n g an i n t e r n a l r a d i o a c t i v e g l u t a m a t e r e s i d u e when c o m p a r e d w i t h t h e t e r m i n a l l y L?"^C]-1abeiied short chain substrate ( F i g . 4).

F i g . 4. The p r e f e r e n t i a l c l e a v a g e o f l o n g e r c h a i n pteroylpolyglutamates. The r e l e a s e o f r a d i o a c t i v i t y f r o m P t e - G l u - G l u - 0 - 4 ( 3 - G l u ( # ) , 10 uM, P t e - G l u - I 1 4 c j G l u - G l u - G l u - G l u ( • ) , 10 ^iM and a m i x t u r e o f b o t h o f

90 t h e s e substrates (•), each 10 p M , is shown a function of t h e t i m e of incubation.

as

M i x t u r e s of t h e s e two substrates gave a p a t t e r n of release of radioactivity similar to that for t h e longer chain substrate indicating it.s preferential selection. (c) Bond cleavage is independent of t h e N H 2 - t e r m i n a l pteroyl moeity. The free polyglutamate (Glu-Glu-Glu) w a s cleaved almost at t h e same rate as t h e Pte-Glu-Glu-Glu while t h e NH2~terminal substituted t - B o c derivative w a s h y d r o l y s e d more r a p i d l y , an effect which might be related to t h e t o t a l net charge of t h e substrate (Fig. 5).

Fig. 5The release of radioactivity from Boc-Glu-_. L 1 4 C ] - G 1 U - G 1 U ( » ) } P t e - G l u - L 1 4 C J - G l u ( o ) and Glu-[l4cJG l u - G l u ( A ) by conjugase as a function of time. (d) The e n z y m e , as might be expected from what has b e e n discussed above, is a hydrolase not a carboxypeptidase. It will attack almost any gamma bond at random although it seems to p r e f e r internal bonds. T h i s is reflected in t h e chain length of t h e v a r i o u s p e p t i d e s released during a t i m e course study of t h e action of the enzyme on Pte-Glu-Glu—GluG l u - L 1 4 C j - G l u (Fig. 6).

F i g . 6. The r e l a t i v e a m o u n t s o f o r i g i n a l enzyme s u b s t r a t e ( P t e - G l u - G l u - G l u - G l u - L 1 ^ - G l u ) ( • ) , and glutamate d e r i v a t i v e s (expressed as a percentage o f t h e t o t a l a s s a y 14C r a d i o a c t i v i t y ) p r e s e n t d u r i n g t h e c o u r s e of e n z y m a t i c h y d r o l y s i s . The v a r i o u s d e r i v a t i v e s are tetraglutamate ( A ) , triglutamate ( • ) , d i g l u t a m a t e ( o ) and g l u t a m i c a c i d ( A ) . The e n d p r o d u c t s of t h e a c t i o n o f t h e enzyme a r e p t e r o y l g l u t a m i c a c i d and g l u t a m i c a c i d . A detailed report of t h e s e s t u d i e s i s i n p r e s s i n t h e J o u r n a l of B i o l o g i c a l C h e m i s t r y i n J u l y / A u g u s t , 19 7 5 . ACKNOWLEDGEMENTS T h e s e s t u d i e s w e r e s u p p o r t e d by g r a n t s f r o m t h e N a t i o n a l H e a l t h and M e d i c a l R e s e a r c h C o u n c i l of A u s t r a l i a and t h e New S o u t h W a l e s S t a t e C a n c e r C o u n c i l . REFERENCES 1. Kovacs, J . , Kapoor, A . , Ghatack, U . R . , M a y e r s , G . L . , G i a n n a s i o , V.R. , G i a n n o t t i , R . , S e n y k , G . , N i t e c k i , D.E. a n d Goodman, J . W . B i o c h e m i s t r y 11_, I 9 5 3 - I 9 5 8 ( I 9 7 2 ) . 2.

B a u g h , C.M. a n d K r u m d i e c k , 186, 7-28 (1971).

3.

S i l i n k , M., R e d d e l , R . R . , B e t h e l , J . B i o l . Chem. i n p r e s s J u l y / A u g .

4.

Silink,

5.

A b r a m o w i t z , N . , S c h e c t e r , I . and B e r g e r , B i o p h y s . R e s . Comm. 862-867 ( 1 9 6 7 ) .

M. and Rowe, P . B .

28-36 ( 1 9 7 5 ) .

C.L,

Ann. N.Y. A c a d . M. and Rowe, (1975).

Biochim.

Biophys. A,

Sci.

P.B.

Acta.

381 ,

Biochem.

92 DISCUSSION Cossins: The enzyme does not require the pteridine moiety for activity. Is it therefore a folate hydrolase ? Rowe: No. This is the reason the name y-glutamyl hydrolase has been proposed as a definitive assignation. Mautner: Can your enzyme be protected against inhibition by PCMB by adding substrate ? Rowe: No. The inhibition is potent and instantaneous. We have not, however, explored this possibility in any detail, i.e. over a wide range of concentration. Mautner: The fact that PCMB inhibits doesn't prove that one deals with a thiol enzyme (f.i. aspartate transcarbamylase irihib. by PCMB, but no SH in cat. site). Rowe: Agreed, but the enzyme is very sensitive to heavy metals also. The effect of PCMB must be related to the active site as the binding stabilises the activity virtually indefinitively, i.e. the effect can be reversed after many months. The requirement for 5.o mm i3-mercaptoethanol for stabilisation would also suggest that -SH groups are important. Kisliuk: What is the effect of NaCl concentration on the activity of yglutamyl hydrolase ? Rowe: The enzyme is particularly sensitive to the effects of anions, which is probably at least partly related to the polyamide nature of the substrate. Na + ions produced no effect on the enzyme up to 5o mm concentration. 0.1 M NaCl produced 5o I inhibition. The question is quite complex and I refer you to the material to be published in the 'Journal of Biological Chemistry' in August 1975. Cooper: Is the conjugase in the plasma the same enzyme, and do you think that it is derived from liver lysozomal enzyme ? Rowe: I don't know at least for human serum or beef serum. The pH optima for both of these enzymes is higher. We have not characterized any of the serum enzymes. It would be interesting to know particularly whether there are glycoproteins. There are implications, of course, in the question that are important to consider when one is using crude tissue preparations involving polyglutamate derivatives. Scrimgeour: The pH optimum of 3.9 is intriguing, along with the drastic drop in activity above pH 4.5. The optimum is near the pH of lowest solubility of folates. Could this relationship have some subtle type of control function ? Rowe: It is possible, but I don't really know about the solubility control unless I had figures on the intracellular concentration of the pteroyl polyglutamates. Furthermore, it is quite possible that the in-vivo pH-optimum is quite different. For example changing the assay buffer from 33 mm acetate to 33 mm citrate changes the pH-optimum from 3.9 to 4.5 with quite a different profile.

The Purification of Riboflavin Synthetase by Affinity Chromatography Using 7-Oxolumazines ft Wrigglesworth, C.D. Ginger, R.J. Kulick and H.C.S. Wood

R i b o f l a v i n synthetase i s an enzyme found in various microorganisms which synthesises one molecule of the vitamin, r i b o f l a v i n , from two molecules of the enzymic substrate 6 , 7 - d i m e t h y l - 8 - D - r i b i t y l l u m a z i n e compound").

The other product of the reaction i s

("G

4-£-ribitylamino-5-

aminouracil.

O

O

•NH2

+

CH,

1 H

CH,

I

J

2

NH

I

2

(pHOH)3

(CHOH)O

(CHOH)O

CH 2 OH

CH 2 OH

CH 2 OH

"G compound"

I

J

I

3

Riboflavin

A mechanism has been proposed for t h i s reaction based on n.m.r. monitored deuterium l a b e l l i n g experiments in a non-enzymic model system (1) and more recently t h i s proposal has been substantiated in i t s e s s e n t i a l aspects by work on the corresponding enzymic system (2).

94

The stoichiometry of the reaction requires that a four carbon moiety be transferred from one lumazine molecule to the other and i t has been proposed that an early step in the sequence involves nucleophilic attack by the charge d e l o c a l i s e d anion of the C^-acceptor lumazine on the modified C^-donor.

Further steps involve r i n g opening of the tetrahydropyrazine

ring of the donor lumazine to allow the t r a n s f e r of the C^ u n i t , and f i n a l l y c y c l i s a t i o n to give r i b o f l a v i n . With a view to further i n v e s t i g a t i o n of the enzymic r e a c t i o n , in p a r t i c u l a r to locate and i d e n t i f y the proposed enzyme nucleophile X, a programme aimed at p u r i f i c a t i o n of the enzyme for a c t i v e - s i t e studies was embarked upon.

labelling

95 According to the transition state analogue hypothesis (3) it is to be expected that compounds similar in structure to the C^-acceptor lumazine anion would bind strongly to riboflavin synthetase.

The observation that

the naturally occurring 6-methyl-7-oxo-8-D-ribitynumazine, (I, R = Me), (4), is a good competitive inhibitor of the enzyme, (5), provides support for this hypothesis. A series of 7-oxo-8-D-ribityllumazines have been synthesised by the reaction sequence shown below where 4-D-ribitylamino-5-nitrouracil

is

catalytically reduced to the unstable diaminouraci1 which is condensed with the relevant a-keto acid (or ester) under acid conditions to give the series of oxolumazines (I).

The inhibition constants (Ki) show this

series of compounds to be potent inhibitors of riboflavin synthetase.

O

H

O

H

D-Ribityl

D-Ribityl

1

Ki (M)

I

R = Me Et CH 2 PH COOH CH2CH2-COOH

I

D-Ribityl

96 The compound I (R = -CH^.CH^-COOH) was specifically designed for use as a ligand in affinity chromatography and has been coupled to Sepharose 4B by the route outlined below.

Sepharose 4B was activated using cyanogen bromide (6) to give the postulated iminocarbonate derivative.

This was reacted with the "spacer

arm", 4-azaheptane-l,7-diamine to give the amino-functionalised

polymer

which was then attached to the oxolumazine by a water-soluble carbodiimide mediated condensation reaction at pH 6.7.

The polymeric product exhibited bright blue fluorescence, a characteristic of the 7-oxo-8-ID-ribityl lumazines.

It was further

characterised by its ultraviolet absorption spectrum measured as a suspension and after aqueous hydrolysis.

The spectrum of the immobilised

ligarid ( ^ m a x pH 1, 329, 281 nm) was directly superimposable on that of the free ligand.

By this method a coupling of 1.2 umoles of 7-oxolumazine per

ml. of settled polymer was calculated.

— OH

CNBr

Agarose

— O •» A g a r o s e

— OH

— O

NH 2 (CH 2 )} 3 NH(CH 22) 33NH. 2 2 2> — OH Agarose

I(R=(CH9)9.COOH) Agarose

— OCONH(CH J9 ).NH 23

— OH

— OCONH(CH29J)^NH 3

O

NH.

2

D-Ribityl

97 Coupling of the ligand at pH 4.6 instead of 6.7 gave an immobilised product which showed a u.v. spectrum ( * m a x pH 1, 270 nm) very d i f f e r e n t from free 7-oxolumazine.

This material was e f f e c t i v e as an a f f i n i t y

chromatography support for r i b o f l a v i n synthetase i n a s i m i l a r manner to the material in the experiment described below.

The structure of t h i s

material and a product obtained from a related model reaction in free s o l u t i o n are c u r r e n t l y under i n v e s t i g a t i o n . A p p l i c a t i o n of a p a r t i a l l y p u r i f i e d extract of r i b o f l a v i n

synthetase

from bakers yeast to a column of the immobilised 7-oxolumazine resulted in complete retention of a c t i v i t y whereas a large amount of i n a c t i v e protein passed through in the void volume of the column.

Washing of the column

with 0.5 M potassium chloride removed more i n a c t i v e protein which had been retained in the column by non-specific i o n i c binding. was s p e c i f i c a l l y eluted from the column using 10 f r a c t i o n s being assayed d i r e c t l y on e l u t i o n .

The active enzyme

M substrate, the

Subsequent exhaustive

d i a l y s i s of the pooled active f r a c t i o n s removed the substrate with concomitant l o s s of enzymic a c t i v i t y .

This process however allowed an

estimate of protein concentration to be made and hence the s p e c i f i c a c t i v i t y of the eluted enzyme was calculated.

Affinity Column Elution P r o f i l e

Elution volume A

-

e l u t i o n w i t h 0 . 5 M KC1

B

-

e l u t i o n w i t h 10

-2

M substrate

98 Purifications of riboflavin syntheta se of the order of 1000 fold with recoveries of 60 - 70% of active material were thus achieved, which exemplify the usefulness of affinity chromatography relative to the more classical methods of enzymic purification.

With development this method

should allow large quantities of purified enzyme to be obtained and characterised for use in further studies of the active-site. ACKNOWLEDGEMENTS We thank the S.R.C. for a Research Grant and the Distillers Company Limited for generous gifts of dried bakers yeast. REFERENCES 1.

Paterson, T. and Wood, H. C. S. Part VI.

The Biosynthesis of Pteridines.

Studies of the Mechanism of Riboflavin Biosynthesis.

J. Chem. Soc., Perk I, 1051 - 1056 (1972);

Deuterium Exchange of

C-Methyl Protons in 6,7-Dimethyl-8-D-ribityllumazine, and Studies of the Mechanism of Riboflavin Biosynthesis.

J. Chem. Soc., Chem. Comm.,

290 - 291 (1969). 2.

Beach, R. L. and Plaut, G. W. E.

Stereospecificity of the Enzymic

Synthesis of the o-Xylene Ring of Riboflavin.

J. Amer. Chem. Soc.,

92, 2913 - 2916 (1970). 3.

Wolfenden, R.

Analog Approaches to the Structure of the Transition

State in Enzymic Reactions.

Accounts of Chem. Research. _5, 10 - 17

(1972). 4.

Masuda, T. ashbyii.

5.

Isolation of Some New Substances Produced by Eremothecium Chem. Pharm. Bull. Tokyo;

4, 72 - 74 (1956).

Winestock, C. H., Aogaichi, T. and Plaut, G. W. E. Specificity of Riboflavin Synthetase.

The Substrate

J. Biol. Chem., 238, 2866 -

2874 (1963). 6.

Cuatrecasas, P.

Protein Purification by Affinity Chromatography.

J. Biol. Chem., 245, 3059 - 3065 (1970).

99 DISCUSSION Whiteley: Why was the particular spacer group used to attach the ligand to the active Sepharose ? Wrigglesworth: To avoid non-specific hydrophobic binding. The recent literature has implied that many of the early "affinity chromatography" purifications were in fact due to hydrophobic binding effects and we used this more hydrophilic spacer arm to eradicate these. Scrimgeour: Do you have any estimate of the purity of your enzyme after the one-step purification ? Wrigglesworth: Preliminary page experiments showed only one band on the gel. McCormack: What degree of potency and what type of inhibition (e.g. competitive or non-competitive) was observed with the 7-oxo-8-ribityl-lumazine bearing hydrogen at position 6 ? Wrigglesworth: We have made this compound but were unable to purify it completely and consequently we have no quantitative inhibition data.

Interaction of Riboflavin Synthetase with Analogues of 6,7-Dimethyl-8-Ribityllumazine G.W.E. Plaut and R.L. Beach

ABSTRACT Nine analogues of 6,7-dimethyl-8-D-ribityllumazine

substituted

at position 8 w i t h D - and JL-threityl, ])- and L-erythrityl, DL-glycerityl, 3'-hydroxypropyl, 2'-deoxy-I)-ribityl, 3'-dexoy-D-ribityl,

4'-deoxy-D-

- r i b i t y l chains w e r e tested for kinetic activity w i t h purified yeast riboflavin synthetase.

None of the compounds w a s a substrate; however,

the L - t h r e i t y l and 3'-deoxy-D-ribityl analogues w e r e inhibitors

compe-

titive w i t h 6,7-dimethyl-8-D-ribityllumazine w i t h K i = 190 p M and 1.2 yM, respectively. T

7ith the synthesis of 6,7-dimethy 1 - 8 - [1' (4 '-deoxy-D-ribityl) ]lumazine

all of the mono-deoxy-D-ribityl analogues have n o w b e e n prepared. A further consideration of the enzymic m e c h a n i s m for the conversion of 6,7-dimethyl-8-D-ribityllumazine CPK space filling models.

to riboflavin is presented using

It is proposed that two different

substrate

conformers may interact at the donor and at the acceptor sites of the enzyme.

Furthermore, similarities in the structures of the natural

substrate and of analogues exhibiting k i n e t i c activity are compared and contrasted w i t h conformations of some of the kinetically inactive analogues.

The comparisons are b a s e d on the effect of the rela-

tive positions of the 2' and 3* hydroxyl groups on gross structure.

102

It has been shown in a number of laboratories that the last step in riboflavin biosynthesis involves a condensation between two molecules of 6,7-dimethyl-8-ribityllumazine to form one molecule each of riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine (for review see Plaut (1971), Demain (1972), Plaut et al. (1974)).

Ribo-

flavin synthetase, the enzyme which catalyzes the reaction, has been detected in a larger number of microorganisms and in plants.

It has

been purified 5000-fold from bakers yeast extract, the best preparations exhibiting a single zone of protein possessing the activity when examined by cellulose acetate and polyacrylamide disc gel electrophoresis (Plaut & Harvey, 1971). As shown in Fig. 1 the reaction involves a transfer from one molecule of 6,7-dimethyl-8-ribityllumazine to a second molecule of the lumazine of a 4-carbon moiety containing carbons 6 and 7 and the attached methyl groups.

In this transfer, the methyl groups attached

to carbons 6 and 7 of the lumazine accepting the 4-carbon moiety are 0

0

Ribityl

Ribityl Acceptor

Donor

1

1 H

0

Ribityl

H

0

Ribityl

Figure 1: Conversion of 2 molecules of 6,7-dimethyl-8-ribityllumazine to riboflavin (2) and 4-ribitylamino-5-amino-2,6-dioxopyrimidine (3).

recovered in carbons 5 and 8 of riboflavin, respectively, whereas the m e t h y l groups attached to carbons 6 and 7 of the A-carbon donor lumazine become the m e t h y l groups at carbons 7 and 6 of riboflavin, respectively

(Beach & Plaut, 1970b).

It is significant that in the

chemical formation of flavins from such lumazines, w h i c h occur under neutral (Rowan & Wood, 1963, 1968) and acid conditions (Beach & Plaut 1969), the same orientation has b e e n observed of the carbons derived from the 6,7-dimethyl lumazines to form the o-xylene ring of flavin (Paterson & W o o d , 1969).

This may suggest that the orientation of

the 4-carbon piece transferred in riboflavin formation is determined b y the inherent chemical properties of the lumazine m o l e c u l e rather than b y being directed by the stereochemical effects of enzyme cataly sis. Chemical experiments showed that in I^O the protium of the 7 - m e t h y l (but not the 6-methyl) of 6,7-dimethyl-8-aldityllumazines replaced by deuterium

(Table I).

is

The exchange at the 7-methyl group

occurred more rapidly under alkaline and a c i d conditions than at neutrality

(Paterson & W o o d , 1969; Stewart & McAndless, 1970; Beach

& Plaut, 1969, 1970a).

These observations m a d e it possible to test

(a) w h e t h e r replacement of H by D at the 6-methyl or 7-methyl groups has a n effect on the enzymic transformation of the substrate to p r o duct and (b) whether the exchange reaction at the 7-methyl group is catalyzed b y the enzyme and, therefore, is an intrinsic part of the m e c h a n i s m of the overall reaction.

The enzymatic transformation of

6-methyl-7-deuteromethyl-8-ribityllumazine

to flavin proceeded at

about 80% of the velocity of the non-labeled substrate.

However, a

very substantial isotope effect w a s observed w h e n the transformation

104

m 1 CSI ' 1 O •t *—V sí -—' 1 UM

oa O Ci •Ö G M

•O m tu w •J pa *H cMmincocMcov£>cM m vo tu co eofl oi OHÄ IH 0) 4J TP -H tí UH IU JP C M A « «t fi A —' * — '

t-l 00 co 1 O• 00• sr o

l-l ^ Jp 4-1 tu X

i-H ^ X 4-1 tu X

oX [0 ,—«co^ — . Pi en P3 m ••—' ^

^

•—' ^—'

1 cu \o tí rH •H 4-1 Xu CH N CO •H X 4-1 O CO S •a •-I 4-1 •H i-I 3 p o 4-1 4-i o co CO rtí •H •H rH -C ¡> 4J +J ¿J !>> ^ •H CU CO •H X P3 l-l e X CO 4-1 1 CU •H 3 O CU O P Ul C-, S tM

>< >s rH U U U >i -H iH -H O.H Í Í O "ri H H K U h (J M p-, >, ftTt 1,1,1, ^ ÍS'rt rlJCO H M• X nS;>,00' ' •H iH JP JP 4-1 4-> l-l >s " o o tu ÍU 4J 4-1 -H vH t rH O tu tu H H >> ^ ^ H ï ^ o (U .C J-1 -H PQ l , D o p I E-i H W H & X! I iJ I I. I, I. I. I.I P hj C hi n e n e « Cn

B cO X

tu Oil tí CO X O X tu CO O. tu tí 6 Jp co co H O 3 4-1 co •H -H m 4-1 H •H •o ra cu CU & U 4J 4P 3 co CO 4-1 OL VO h OP. s OC 4-1 tu X O Jp V4 MH U tícO o co O •o cu 4-1 UH •o 4-1 tí Jp CO O C4H •H tí g •H B 4-1 O O 4-1 X ti tí 4-1 cu co títu cl-lu ai 4-J U bt CU rH 4J 4-J rH tu -H 4-1 E. 3 tí CO •C MH tu •H P. tu tu tíOC 4-1 efl O •H rH rH •H rH rH •H l-i •H CL, C X P. 4-1 b. P. CO S&AcH3(.287ppin)

J 0H2

H F

H

CH3 (-2.17PP„J

0

CH3 (-1.37,,,») 2,0

1,0

S •s

ea 4

5 6 7 Days m culture

Fig. 3 . Activity of serine hydroxymethyltransferase during the c u l ture cycle of MF and L - c e l l s was assayed at 37 C in a t o t a l volume of 1 . 2 ml with 4l.5,umoles of glycine, 2.0.umoles of formaldehyde, 0 . 5 umole of tetrahyttrofolate, 2 . 6 nmoles ox pyridoxal phosphate, 6.25 iimoles of potassium phosphate buffer, pH 7 . 5 and enzyme e x t r a c t Corresponding t o 120-400 ,ug of protein. Each value represents the mean of at l e a s t 5 separate estimations + SD. • -MF; 4 - L - c e l l s .

147 o. 5, 10-methylenetetrahydrofolate dehydrogenase. The a c t i v i t y of t h i s enzyme in e x t r a c t s both of MF and L - c e l l s was nearly constant during the culture c y c l e , although i t s level in L - c e l l s was about twice of that found in MF ( F i g . 4 ) . 60

c S "o 50 ts.

40

6—. i * 30 g ir> 20 TS i

10

1

2

3

4

5

6

7

Days in culture

Fig. Activity of 5,10-methylenetetrahydrofolate dehydrogegase during the culture cycle of MF and L - c e l l s was assayed at 37 C in a t o t a l volume of 1 ml: 0.6,umole of tetrahydrofolate, 0.4,umole of NADP, 1,umol of pyridoxal phosphate, 2.5/Umoles of cysteine, 3 . 6 umoles of s e r i n e , 3«0/Umoles of KHCO-., 50/Umoles of potassium phosphat e buffer, pH 7 . 5 and enzyme extract^corresponding to 100-350 ,ug of protein. Each value represents the mean of at l e a s t 5 separate estimations + SD. • -MF; A - L - c e l l s . d. Formyltetrahydrofolate synthetase. I t s a c t i v i t y in e x t r a c t s from mouse or human f i b r o b l a s t s and from mouse or human transformed c e l l s (13) was always higher in the transformed c e l l s , but of the same level f o r the particular type of c e l l s when tested from p r o l i f e r a t i n g and apparently contact inhibited c e l l s ( i . e . a f t e r two and four to s i x days a f t e r subculturing, r e s p e c t i v e l y ) . The data now presented ( F i g . 5 ) confirmed these findings. However, the formyltetrahydrofolate synthetase of MF when assayed soon a f t e r subculturing (1 day) was the highest and decreased during two subsequent days in culture and was subsequently constant. Thus, both patterns of the a c t i v i t y curves of formyltetrahydrofolate synthetase of MF and L - c e l l s d i f f e r from those found f o r human skin f i b r o b l a s t s (8) and f o r the lymphoblastoid human c e l l line R.P.M.I. 4265 ( 2 0 ) .

148 Fig. 5. Activity of formyltetrahydrofolate synthetase during the culture cycle of ^F and L-cells was assayed at 37 C in a total volume of 1 ml: 40-umoles of formate, 0.66/umole of tetrahydrofolate, 0.b6,umole of ATP, 4/Umoles of cysteine, 2.6,umoles of MgClp, 33/Umoles of NH4C1, 100 moles of Tris-HCl buffer at pH .0 and enzyme extract corresponding to about 200 ,ug of protein. Each value represents the mean of at least 4 separate estimations ± SD. • - M F } A —L-cells.

1 2

3

4

5

6

7 Oiyj

DISCUSSION In this report the activities of four folate-metabolizing enzymes of mouse fibroblasts (MF) and L-cells were compared as determined in extracts from cells harvested at subsequent days of the culture cycle. Even though the cell populations were not synchronized under conditions of our experiments, the levels of enzymic activities in cell extracts are believed to be representative to some extent of the enzyme activities within a standard cell of our populations. The enzymes under investigations were: dihydrofolate reductase, serine hydroxymethyltransferase, methylenetetrahydrofolate dehydrogenase and formyltetrahydrofolate synthetase. The data presented here showed that the activities of all four enzymes of L-cells throughout the culture tfycle were considerably higher than those of MF. Of all the four enzymes tested only dihydrofolate reductase was found to be an enzyme typical for the logarithmic phase of growth both for MF and L-cells. Thus, after an initial increase in the activity of the enzyme, a subsequent decrease of its activity was coincident with a drastic fall in mitotic activity of both types of cells when confluence population density was reached. It is noteworthy however, that while the activity of dihydrofolate reductase of MF approached zero, the minimum activity of this enzyme of L-cells

149

was still as high as the maximum activity of MF, even though mitotic-indices both for MF and L-cells were nearly zero. This means that dihydrofolate reductase activity in L-cells persists at a quite high level even when these cells cease to divide. This former phenomenon is also consistent with our previous finding that dihydrofolate reductase activity sustained at high although variable level in cells of several transformed lines, including L-cells (13). The variations of the activity of this enzyme from the transformed cells were interpreteted in terms of the existence of the enzyme in different conformational states with different activities. We wonder whether dihydrofolate reductase of L-cells from the confluent culture actually is in its activated state. The high activity of dihydrofolate reductase in extracts of MF and L-cells from the proliferating cultures as compared with that from the stationary ones might suggest that the tetrahydrofolate pool in these cells may differ. This in turn might influence to some extent the levels of the enzymes utilizing tetrahydrofolate as one of the substrates. However, the level of activity of formyltetrahydrofolate synthetase decreased only slightly during the culture cycle, whereas those of methylenetetrahydrofolate dehydrogenase of MF and L-cells as well as that of serine hydroxymethyltransferase of MF were nearly constant. The relatively high but transitory increase in the activity of serine hydroxymethyltransferase of L-cells, simultaneously with drastic fall of dihydrofolate reductase and mitotic activities is difficult to interprete. It might be, however, related with the intensity of methionine biosynthesis which seems to be more active in L-cells than in mouse fibroblasts (preliminary data from this laboratory). REFERENCES 1. Kit,S., Dubs,D.R., Frearson,P.M.:Decline of thymidine kinase activity in stationary phase mouse fibroblast cells. J. Biol. Chem. 240. 2565-2573 (1965). 2. Eker,P.: Studies on thymidine kinase, thymidylate kinase and deoxycytidylate deaminase of Chang liver cells. J. Biol. Chem. 243, 1979-1984 (1968). 3. Littlefield,J.W.: Studies on thymidine kinase in cultured mouse fibroblasts. Biochim. Biophys. Acta 14-22 (1965).

150 4. Hooper,A.V:: Thymidylate synthetase in mammalian cells in vitro. J. Cell Biol. ¿ , 59A (196?). 5. Rosenberg,R.N., Vandeventer,L., de Francesco,L., Friedkin,M.E.s Regulation of the synthesis of choline-O-acetyltransferase and thymidylate synthetase in mouse neuroblastoma in cell culture. Proc. Nat. Acad. Sci. USA 68, 1436-1440 (1971). 6. Conrad,A.H.: Thymidylate synthetase activity in culture mamalian cells. J. Biol. Chem. 246, 1318-1323 (1971). 7. Conrad,A.H., Ruddle,F.H.s Regulation of thymidylate synthetase activity in cultured mammalian cells. J. Cell.Sci. 10, 471-486 (1972). 8. Rosenblatt,D.S., Erbe,R.W.: Reciprocal changes in the levels of functionally related folate enzymes during the culture cycle in human fibroblasts. Biochem. Biophys. Res. Commun. ¿4, 1627-1633 (1973). 9. Chang,L.M.S., Brown,M.f Bollum,F.J.: Induction of DNA polymerase in mouse L cells. J. Mol. Biol. 74, 1-8 (1973). 10.Horvat,A., Acs,6.: Inductionof lysosomal enzymes in contact inhibited 3T3 cells. J. Cell. Physiol. 8^, 59-68 (1974). 11.HillcoatjB.L., Swett.V., Bertino,J.R.: Increase of dihydrofolate reductase activity in cultured mammalian oells after exposure to methotrexate. Proc. Nat. Acad. Sci. USA ¿8, 1632-1637 (1967). 12.Jackson,R.C., Huennekens,F.M.: Turnover of dihydrofolate reductase in rapidly dividing cells. Arch. Biochem. Biophys. 154. 192-198 (1973). 13.Zielinska,Z.M., Grzelakowska-Sztabert,B., Koziorowska,J., Manteuffel-Cymborowska,M.: Dihydrofolate reductase and formyltetrahydrofolate synthetase of mammalian oells of different characteristics. Int. J. Biochem. 173-182 (1974). l4.Scrimgeour,K.G., Huennekens,F.M.s Folic acid-dependent enzymes involved in one-carbon metabolism. In Handbuch der Physiologisch-Chemischen Analyse 68, 181-208, Berlin, Springer Verlag (1966). 15.Hatefi,Y., Osborn,M.J., Kay.L.D., Huenriekens,F.M.: Hydroxymethyl tetrahydrofolic dehydrogenase. J. Biol. Chem. 227. 637-64? (1957). 16.Lowry,0.H., Rosebrough,N.J., Farr,A.L., Randall,K.J.: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193. 265-275 (1951). 17.Futterman,S.: Enzymatic reduction of folic acid and dihydrofolic acid to tetrahydrofolic acid. J. Biol. Chem. 228, 1031-1038 (1957). 18.Blakley,R.L.: Crystalline dihydropteroylglutamic acid. Nature (London) 188, 231-232 (I960). 19.Zakrzewski,S.F., Sansone,A.M.: A new preparation of tetrahydrofolic acid. In Methods in Enzymology (ed.Colowick and Kaplan) 18, 728-731, New York, Academic Press (1971). 20.Chello,P.L., Bertino,J.R.: Effect of carboxypeptidase G. on levels of dihydrofolate reductase and N,.- formyltetrahyarofolate synthetase in human 4265 leukemia cells In tissue culture. Proc. Am. Ass. Cancer Res. abstr. 353, P. 89 (1972).

151 DISCUSSION Poe: Could you give the exact details of your dihydrofolate assays and how you correct for NADPH-oxidase ? We have discovered the NADPH-oxidase of mammalian sources (pig, monkey, mouse) is stimulated by 2-mercaptoethanol. Grzelakowska-Sztabert: The activity of dihydrofolate reductase was determined by a spectrophotometric method based on the decrease in absorbance at 34o nm caused by the enzymatic oxidation of NADPH. The reaction was started by the addition of dihydrofolate to the incubation mixture which has been standing at least 5 minutes. I admit we were not aware of the fact that in our experimental system NADPH-oxidase may be active. Thus I can only hope that the preincubation period of the incubation mixture without dihydrofolate was sufficiently long for the reaction catalysed by NADPHoxidase to be completed before we started the measurements of dihydrofoLate reductase activity. Of course, it is obvious now that we have to check w h e therNADPH-oxidase was active in the cell extracts studied by us. As a matter of fact we used enzyme extracts already devoid of the proteins precipitating at pH 5.1; thus it also may be possible that NADPH-oxidase was among these proteins. Huennekens: Are your cells grown on folate and what is the level and type of screen used ? Have you used 5-Methyl-tetrahydrofolate as a folate source ? Grzelakowska-Sztabert: Either mouse fibroblasts or L-cells were grown as monolayers in Eagle's minimum essential medium (MEM) supplemented with 104 heat inactivated calf serum, L-glutamine (final concentration 2 x 10"3 m) and antibiotics. This medium contains folate at concentration 1 mg/boo ml. up to now we have not used 5-methyl-tetrahydrofolate as a folate source, but I think it may influence to great extent the activity of some folatemetabolizing enzymes, especially those involved in the methionine biosynthesis.

The Activity of the Cobalamine-dependent MethionineSynthetase (5-Methyl-5, 6, 7, 8-Tetrahydrofolic Acid: Homocysteine Methyltransferase) in Rapidly Growing Human Cells and the Effect of Some Folic Acid Derivatives and Analogues H. Sauer and W. Wilmanns Summary: With regard, to the defective cell maturation and proliferation in the case of folic acid or vitamine B12 deficiency methionine-synthetase (MS) is one of the most interesting enzymes because of its dependency on both vitamines. MS was found to be directly correlated to the proliferation tendency of cultured human lymphoblasts• According to this results there are high enzyme activities in leukemic cells and low activities in bone marrow cells of patients with pernicious anemia. Folic acid derivatives and analogues have no direct inhibiting nor stimulating effect on MS activity neither in vitro nor in cultured cells. A n active MS is needed for the rescue effect of 5-methyltetrahydrofolate and 5-formyl-tetrahydrofolate after dihydrofolate-reductase inhibition with amethopterine. Abbreviations: THF 5-CH3-THF 10-CH0-THF 5-IO-CH2-THF 5-10-CH-THF DNA-P MS TK

= = = = = = = =

5,6 ,7,8-tetrahydrofolic acid (monoglutamate) 5-methyl-5,6,7,8-tetrahydrofolic acid 5-formyl-5,6,7,8-tetrahydrofolic acid 5-10-methylen-tetrahydrofolic acid 5-10-methenyl-5,6,7,8-tetrahydrofolic acid Deoxyribonucleic acid polymerase Methionine-synthetase Thymidine-kinase.

Acknowledgements: 5-CH3-THF was a gift from Prof.Dr. L. Jaenicke, Inst, of Biol.Chem., Univ. of Cologne, West Germany. R 5-CHO-THF (Leucovorin ) and amethopterine (Methotrexat ) was a gift from Lederle Laboratories, Cyanamid GmbH, Munic, WestGermany . Supported by the DEUTSCHE FORSCHUNGSGEMEINSCHAFT.

154 In c l i n i c a l h e m a t o l o g y we are c o n f r o n t e d w i t h

the m e g a l o -

b l a s t i c a n e m i a s i n case of v i t a m i n e B 1 2 and./or folic a c i d f i c i e n c y . It is w e l l k n o w n , that i n s u c h a d e f e c t i v e there i s an i n h i b i t i o n

de-

status

of the p r o l i f e r a t i o n a n d m a t u r a t i o n of

r a p i d l y g r o w i n g c e l l s y s t e m s . In the case of folate

deficiency

it is r a t h e r clear, that the l a c k of folate c a u s e s a n i m p a i r m e n t of the de n o v o s y n t h e s i s of D N A - p r e c u r s o r s . The

diminuation

of the p o o l of " f o l a t e - a c t i v a t e d " o n e - c a r b o n - u n i t s a f f e c t s

the

b i o c h e m i c a l p a t h w a y of the s y n t h e s i s of p u r i n e s e n d p y r i m i d i n e s

O n the o t h e r h a n d i n the case of v i t a m i n e B ^

deficiency,

the

b i o c h e m i c a l m e c h a n i s m is s t i l l u n c l e a r . In m a m m a l i a n s y s t e m s w e k n o w o n l y two v i t a m i n e B-)2 d e p e n d e n t e n z y m e s . The i m p a i r m e n t methylmalonyl-CoA-isomerase

a c t i v i t y in v i t a m i n e B ^

i s w e l l studied, h o w e v e r it d o e s n o t s e e m to be r e s p o n s i b l e the d e f e c t i n c e l l p r o l i f e r a t i o n methionine-synthetase

of

deficiency

(/f,26). The o t h e r e n z y m e ,

for the

(MS), i s p e r h a p s the m o r e i n t e r e s t i n g

b e c a u s e of i t s d e p e n d e n c y o n b o t h v i t a m i n e s , B - p a n d folic

one acid

(2). The M S r e a c t i o n c a t a l y z e s the m e t h y l a t i o n of h o m o c y s t e i n e m e t h i o n i n e u s i n g 5 - C H ^ - T H F a s the m e t h y l d o n a t o r .

to

Fig. 1 shows

t h i s r e a c t i o n s c h e m a t i c a l l y . The e n z y m e c o n t a i n s one m o l e c u l e of v i t a m i n e B ^ system

i n i t s a c t i v e c e n t e r , i s d e p e n d e n t on a r e d u c i n g

(i.e. in our in v i t r o s y s t e m d i t h i o e r y t h r o l = C l e a l a n d ' s

reagent) and is activated by S-adenosyl-methionine. Besides

the

f o r m a t i o n of m e t h i o n i n e , the o t h e r a n d m o r e i m p o r t a n t p r o d u c t free T H F , w h i c h i s a g a i n the a c t i v e form of the v i t a m i n e 5-CHj-THF METHIONINE-SYNTHETASE "B|2~ Enzyme" THF Fi£s_l

•

Homocysteine S-Adenosyt-Methionine Reducing System Methionine

folic

is

155 acid, participating in the enzymatic transfer of one-carbonunits, and there especially in the de novo synthesis of purines and pyrimidines (7). Thus T H F is an essential cofactor in the synthesis of DNA-precursors during cell replication. Perhaps the MS or another vitamine B ^

dependent, membrane

fixed

enzyme plays a role in the cell uptake of 5-CH^-THF. In vitamine B ^

deficiency there was found a decreased uptake of 5-CH-j-THF

into human red blood cells and leukocytes

(3»29).

Our present experiments are concerned with the intracellular MS activity. Other authors (21) found a decrease of this enzyme activity during the log-phase growth of cultured human fibroblasts. In contrast to these results, we found that the activity of the MS is directly correlated with the proliferation tendency of a cell population (13,22). As well as thymidine-kinase and DNA-polymerase (2Zf,25) the MS activity increases during the logarithmic growth period of human lymphoblastoid cells in cula) ture f i.e. MS is one of the so-called log-phase-enzymes. The activities of three log-phase-enzymes are presented in Fig. 2. According to this model system, we found high MS activities in rapidly growing human malignant tumors (22) and especially in peripheral and bone marrow cells of patients with acute leukemias (2if,25). For further investigating the importance of an active MS for cell proliferation under experimental conditions of folate and vitamine B ^

depletion we first examined the in vitro effect of

some folic acid derivatives and analogues on this enzyme extracted from our human cultured lymphoblasts. The enzyme was assayed by a modification radioactive

of the method of WEISSBACH et al. (31) with

^CH^-THF and separation of substrate and product

by ion-exchange on Dowex-1

(22). The concentration of 5-CH^-THF

a) The lymphoblastoid cell strain, derived from a patient with infectious mononucleosis,was established and characterized by P D Dr. K. Wilms (Med. Univ. Clinic , Tubingen, W-Germany) (3i+).

156 LOG-PHASE ENZYMES IN GROWING HUMAN LVMPHOBLASTS (MKT)

METHONINE -SYNTHE TASE-ACTIVITY IN BONE MARRCW CEUS nMoLes/mm-1^°cells *s

there is no signifiMETHION INE-SYNTHETASE-ACTIVITY CURING THE GROWTH OF CULTURED HUMAN LYMPH0BLAST5 (MKT)

nMoles / min • l o " eels

0

1

2

•—• Control • — » • CHj-THF i — A • Amethopterine

3

Fig. 5

«

Days

159 cant alteration of the MS activity in the human lymphoblast cultures during the whole growth period after the addition of 5-CH^T H F or amethopterine or both. That means, a good rescue effect is caused by 5-CH^-THF without a significant increment in MS activity. If there really exists the so-called methyl-folate-trap, the activity of MS, normally detectable during the log-phase growth of our cells (see fig. 2), must be high enough to demethylate large amounts of 5-CH^-THF for getting the free coenzyme THF. Because it was found that amethopterine and 5-CH-^-THF are both dependent on the same membrane transport system (9,11,17), there could be another possibility to explain the results. In the presence of high 5-CH-j-THF concentrations in the culture medium, the uptake of amethopterine into the cells may be reduced, so that there is no effective inhibition of the dihydrofolatereductase and no real rescue mechanism takes place. In this case we cannot expect any alteration of the MS activity. Studies, on the intracellular activity of the dihydrofolate-reductase

under

the same culture conditions still have to be done. But first we were going to see, if there is also a rescue effect when the MS activity was inhibited by propyl-iodide (1,2). In METHIONINE-SYNTHETASE-ACTIVITY in vitro Inhibition with ProcM-kxidg (MKT-Ergvme)

IT"

| '/.Activity

J,

VTM

Fig. 6

Propyt-kxSde

160 vitro we found a 50% inhibition of the lymphoblast enzyme with a propyl-iodide concentration of about

M, A dose-response

curve for the propyl-iodide effect on the in vitro MS activity is shown in fig. 6. First results show, that there is no rescue effect when propyliodide (10 ^ M) is added to the culture medium together with amethopterine and 5-CH^-THF , This seems to be an indication, that in our cell cultures the MS reaction plays a special role in this rescue mechanism.

161

R e f e r e n c e s : 1.

Brot, N., Weissbach, H.: Enzymatic synthesis of methionine. J . B i o l . Chem. 240, 3064-3070 (1965).

2.

Burke, G.T., Mangum, J . H . , Brodie, J.D.: Mechanism of mammalian cobalamin-dependent methionine biosynthesis. Biochemistry 10, 3079-3085 (1971).

3.

Chanarin, I . , Perry, J . , Lumb, M.: The biochemical l e s i o n in vitamin-B12 deficiency in man. Lancet, 1251-1252 (1974).

4.

Chanarin, I , England, J . M . , M o l l i n , C., Perry, J . : Methylmalonic acid excretion studies. B r i t . J . Haematol. 25 , 45-53 (1973).

5.

Cohen, S.S.: On the nature of thymineless death. In: Bertino, J . R . (ed.): Folate Antagonists as Chemotherapeutic Agents. Ann. N.Y. Acad. Sei. 186, 292-301 (1971).

6.

Djerassi, I . , Rominger, C . J . , Kim, J . S . , Turchi, J . , Suvansri, U., Hughes, D.: Phase I study of high doses of methotrexate with citrovorum factor in patients with lung cancer. Cancer 30, 22-30 (1972).

7.

Gallo, R.C.: Synthesis and metabolism of DNA and RNA precursors by human normal and leukemic leukocytes. Acta haemat. 45, 136-158 (1971).

8.

Grossowicz, N., Rachmilewith, M., Izak, G.: Absorption of pteroylglutamate and dietary f o l a t e s in man. Amer. J . C l i n . Nutr. 25 , 1135-1139 (1972).

9.

Hoffbrand, A.V., Tripp, E., Catovsky, D., Das, K.C.: Transport of methotrexate into normal haemopoietic c e l l s and into leukaemic c e l l s and i t s e f f e c t s on DNA synthesis. B r i t . J . Haematol. 25, 487-511 (1973).

10. Hryniuk, W., Wolfman, J . , Foerster J . , Bertino, J . R . : Metabolic l i n k between growth rate and c e l l k i l l by methotrexate. In: Gerlach, E. Moser, K., Deutsch, E . , Wilmanns, W.: Erythrocytes, Thrombocytes, Leukocytes. G. Thieme Publishers, Stuttgart, (1973), p. 465-471. 11. Huennekens, F.M., Rader, J . I . , Neef, V., Otting; F . , Jackson, R., Niethammer, D.: Folate antagonists: transport and target s i t e in leukemic c e l l s . In: see 10., p. 496-503 (1973).

162

12. J a f f e , N., F r e i , E . , T r a g g i s , D., Bishop, Y.: Adjuvant methotrexate and citrovorum-factor treatment of osteogenic sarcoma. New Engl.O. Med. 291, 994-997 (1974). 13. J a e n i c k e , L . : The s i g n i f i c a n c e o f methionine synthesis f o r p r o l i f e r a t i o n . In: see 10.p. 362-367 (1973).

cell

14. Jaenicke, L . , Wilmanns, W.: Der Stoffwechsel der Folsäure und der E i n k o h l e n s t o f f e i n h e i t e n . Kl i n . Wschr. 41, 1O29-1O38 (1963). 15. Kutzbach, C . , Stokstad, E . L . R . : Mammalian methylene-tetrahydrofolate reductase. Biochim. Biophys. Acta 250, 459-477 (1971). 16. Mangum, J . H . , Murray, K . B . , North, J . A . : Vitamin B 12 dependent methionine biosynthesis i n c u l t u r e d mammalian c e l l s . Biochemistry 8, 3496-3499 (1969). 17. Niethammer, D., Huennekens, F.M.: Transport of f o l i c a c i d , 5-methyl t e t r a h y d r o f o l i c a c i d and methotrexate through the membrane o f lymphocytes. In: see 10. p. 5o4-5o6 (1973). 18. Nixon, F . P . , B e r t i n o , J . R . : E f f e c t i v e absorption and u t i l i z a t i o n oral t e t r a h y d r o f o l a t e i n man! New Enql. J . Med. 286, 175-179 (1872).

of

19. Rathanasthien, K., B l a i r , O.A., Leeming, R . J . , Cooke, W.T., M e l i k i a n , V.: Folates i n human serum. J . C l i n . P a t h o l . 27, 875-879 (1974). 20. Rosen, G., Suwansirikul, S . , Kwon, C . , Tan, C . , Wu, S . J . , B e a t t i e , E. J . , Murphy, M.L.: High-dose methotrexate with citrovorum f a c t o r and adriamycin i n childhood osteogenic sarcoma. Cancer 33, 1151-1163 (1974). 21. Rosenblatt, D.S., Erbe, R.W.: Reciprocal changes i n the l e v e l o f f u n c t i o n a l l y r e l a t e d f o l a t e enzymes during the c u l t u r e c y c l e in human f i b r o b l a s t s . Biochem. Biophys. Res. Commun. 54, 1627-1633 (1973). 22. Sauer, H., Jaenicke, L . : Einfacher TEST ZUR Messung der MethioninSynthetase ( M S ) - A k t i v i t ä t und seine Anwendungsmöglichkeiten i n der K l i n i k . K l i n . Wschr. 50_, 986-990 (1972). 23. Sauer, H., J a e n i c k e , L . : Zur Aufhebung des z y t o s t a t i s c h e n E f f e k t s von Amethopterin (Methotrexat) durch Methyl-Tetrahydrofolsäure. B l u t 28, 321-326 (1974). 24. Sauer, H., Wilms, K . , Wilmanns, W., J a e n i c k e , L . : Die A k t i v i t ä t der Methionin-Synthetase als P r o l i f e r a t i o n s p a r a m e t e r i n • wachsenden Z e l l e n . Acta Haematol. 49, 200-210 (1973).

163 25. Sauer, H., Wilms, K., Wilmanns, W., Jaenicke, L.: The activity of the methionine-synthetase as an indicator for the proliferation tendency of a cell population. In: see 10.p. 368-371 (1973). 26. Seashore, M.R., Hsia, E., Scully, K., Durant, J.L., Rosenberg,L.E.: Defective propionate oxidation in leukocytes of vitamin B 12-deficient pigs: in vitro correction. Amer. J. Clin. Nutr. 26, 873-875 (1973). 27. Silber, R., Mansouri, A.: Regulation of folate dependent enzymes. In: see 5. p. 55-69 (1971). 28. Tamura, T., Stokstad, E.L.R.: The availability of food folate in man. Brit. J. Haematol. 25, 513-531 (1973). 29. Tisman, G. Herbert, V.: B12 depdendence of cell uptake of serum folate : an explanation of high serum folate and cell folate depletion in B12 deficiency. Blood 41, 465-469 — (1973). . 30. Vogler, W.R., Jacobs, J.: Toxic and therapeutic effects of methotrexate-folinic acid (Leucovorin) in advanced cancer and leukemia. Cancer 28, 894-901 (1971). 31. Weissbach, H., Peterkofsky, A., Redfield, B.G., Dickerman, H.: Studies on the terminal reaction in the biosynthesis of methionine. J. Biol. Chem. 238, 3318-3324 (1963). 32. Whitehead, V.M., Pratt, R., Viallet, A., Cooper, B.A.: Intestinal conversion of folinic acid to 5-methyl-tetrahydrofolate in man. Brit. J. Haematol. 22_, 63-72 (1972). 33. Wilmanns, W.: Unpublished results. 34. Wilms , K.: DNS-Synthese in menschlichen Blutzellen. G. ThiemeVerlag, Copythek, Stuttgart (1975). DISCUSSION Huennekens: In reporting levels of methionine synthetase, do you measure both apo- and holo-forms, or only the latter ? Sauer: We measured only the holo-form of the enzyme. Cyanocobalamineac.etate was added to the assay mixture for completion of the reducing system with Clealand's reagent (Peel-effect; see Peel, J.L.: The catalysis of the auto-oxidation of 2-mercapto-ethanol and other thiols by vitamin B-| 2 derivatives. Biochem. J. 88, 296-3o8 (1963)1.

The Role of Folate Binding Proteins in Folate Metabolism S. Waxman

A new group of folate binding proteins have been described in various tissues and found to be present in several animal species including human.

The purpose of this report will be to review the occurrence,

distribution, characteristics, structure, physiology and possible roles of folate binding proteins in folate metabolism.

OCCURRENCE AND DISTRIBUTION OF FOLATE BINDING PROTEINS The measurement of folate binding proteins may be determined by fractionation of the endogenous protein bound folate activity by gel filtration methods followed by microbiologic assay of each fraction for folate activity (1-5).

Alternatively, the FABP is measured by the binding of

exogenous tritiated pteroylglutamic acid (%PteGlu) to the test substance (8).

At this time there is no study where both endogenous

protein bound folate activity and FABP have been measured simultaneously in a given tissue to determine total folate binding activity.

Attempts

to uncover saturated FABP in serum and milk by removing endogenous "bound" folates by dialysis, charcoal adsorption, acid pH or with 8M urea followed by addition of exogenous %PteGlu have been unsuccessful. This may be due to the irreversibility of the binding of endogenous folate, destruction of folate binding protein by the methods used or that there is only unsaturated FABP in the tissues studied.

Therefore,

at this time it is difficult to definitely relate the endogenous protein-bound folate to the FABP measured by addition of exogenous

166

3HPteGlu.

Endogenous protein bound folate activity has been described in human and various animal tissues by Markkanen and co-workers (1-5) TABLE I. They have found that in human serum almost half of the folate activity is bound to various proteins and the balance is free. bound folate activity was found in three zones.

The protein-

(1) albumin (weakly);

(2) transferrin-y-globulin, and (3) «2-niacroglobulin.

The a2-macro-

globulin appears to have a molecular weight of >150,000, is the most potent folate binder and accounts for 40-45% of the protein bound folate activity in human serum.

The amount of folate bound in the transferrin-

y-globulin fraction appears to increase during pregnancy or when a woman takes oral contraceptives.

They report that when the total serum

folate decreases, as in deficiency states, the proportion of protein bound folate increases.

These authors have described protein-bound

folate in human serum, cerebrospinal fluid, milk and to a lesser amount in the serum of cow, gelding and rabbit. High molecular weight complexes of folic acid have been reported in rat liver, kidney and intestine with molecular weights of 350,000, 150,000, 90,000 and 25,000 (6,7).

Most of these high molecular weight complexes

of folic acid were isolated from the cell supernatant fraction and nuclear fractions and were in the form of folyl polyglutamates. FABP (see TABLE I) has been found in human serum from normals (8), folate deficients (9), some uremics (10), some pregnant women or women taking oral contraceptives (11), milk (12), and in leukocyte lysates from some patients with chronic myelogenous leukemia (13).

FABP has

167 also been reported in bovine (14,15) and goat milk (16), hog kidney (17), the brush border membrane of rat small intestinal epithelial cells (18) and in the serum of the pig (19).

Milk, an apocrine secretion with a

large membrane content, has more FABP than the corresponding serum. Thus, FABP is found in tissues involved in folate transport, utilization or storage.

We have not found FABP in red blood cell ghosts and are

presently assaying,spleen, liver and heart for its presence. TABLE I DISTRIBUTION Or VARIOUS TARP I.

FROTLIN-BOl'ND ENDOGENOUS FOLATE SOURCE

TOTAL FOLATE LEVEL (nf.)

Human Serum

5.2

40-50

" Skim Milk

6.6

60

11.5

5

" Cerebrospinal Fluid Bovine Serum (4) Gelding Serum

M

Hog Serum (4) Sheep Serum

^

Rabbit Serum (4)

II.

% TROTEIN-BOUND FOLATE

^

14.7

11

9.5

10

16.0

21

2.3

46

10.0

11

UNSATURATED FABP ng PteGLU Bound/mg Protein Human Serum (8) Folate Deficient Serum Human Milk

(12)

Bovine Milk

(15)

0.05 0.3 0.7 2.5

Coat Milk < 16 >

7.8

Hog Kidney Extiact (17)

0.4

CHARACTERISTICS OF FABP The characteristics of native FABP have been studied in human serum (9), milk (12), and leukemic leukocyte lysates (13) and share similar characteristics.

Highly purified human (12) (TABLE II), goat (16) and

bovine milk FABP (20,21) have been studied and also share certain

168

characteristics with their native form and with each other.

There is a

rapid association and slow dissociation rate for the binding of 3HPTeGlu. The hog kidney FABP appears to bind %PteGlu irreversibly (22). Binding of PteGlu to FABP occurs over a wide range (pH 5-10) and is maximal from 7.6-10.

There is complete dissociation of PteGlu from FABP at pH Pteroic Acid »tethylH^PteClu MethylH4PteClu( 3 _7)

>MTX

>5 ForinylfypteGlu

DEAE-Cellulose*

Elutes in .001M Phosphate Buffer

Sephadex G-200*

25-40,000 and >100,000

SDS Gel Electrophoresis

87,000, 35.0U0, 19,000

Isoelectric Points

6.8, 7.5, 8.2

Adheres to Con-A Sepharose

(87%) +

PAS Stain MethylH^pteGlu Dissociated by PteGlu* and PteClu( 3 _ 7 ) FABP Bound PteGlu Not a Substrate for DihydroColate Reductase* FABP Bound PteGlu(3_y) Not a Substrate for Conjugase FABP Bound l'teGlu or MethylH^l'teGlu Not Taken Up by Cells

Similar in Serum, llilk, Leukemic Lysates (13) TABLE III

FABP STABILITY EXPERIMENTS

Temperature

-10'C

Addition of PteGlu

Protects

8H Urea

Inhibits

Buffer (at pH 4.8)

>-70°C

>4°C

>23°C

0.1 M Monobasic Polassius Phosphate

32%

0.1 H Monobasic Sodium Phosphate

S.9Z

0.02 M Citrate Buffer

4.9Z

0.1 M Acetate Buffer

4.OX

There is a similar pattern of competition by folate analogs for the binding of ^HPteGlu to various FABP in human and other animal (9).

The preference is for oxidized folyl mono and

tissues

polyglutamates

170 (glutamate chain length 3 to 7) over reduced folyl mono and polyglutamates.

Pteroic acid but not pteridine-6-carboxylic acid or glutamic 3

acid inhibit

HPteGlu binding to FABP.

The presence of a methyl group

in the N^ position diminishes competition whereas a N-* formyl group totally inhibits competition for binding to FABP.

Methyl ItyPTeGlu is

dissociated from FABP by PteGlu or PteGlu(3_7) equally at 23°C or 37°C (23).

Thus, the oxidative state of the pteridine portion and the link-

age to para-amino benzoic acid moiety but not the glutamyl portion are the determinants for the binding of the folate to FABP. COMPOSITION AND STRUCTURE Native human milk and some sera have two distinct peaks of FABP, a >100,000 peak which sediments on centrifugation and a smaller peak at 30-40,000, whereas most human sera and goat milk have only the smaller FABP peak.

The smaller FABP peak in human sera is clearly not trans-

ferrin (24) and in bovine milk is not beta lactoglobulin (21). The smaller FABP from human milk and the goat FABP have been purified from milk using affinity chromatography on PteGlu-Sepharose followed by either sucrose density gradient ultracentrifugation, DEAE-cellulose or isoelectric focusing (12,16) (TABLE IV).

The specific activity of both

FABP was greater than 7 yg PteGlu bound/mg protein.

Purified FABP's are

basic since they elute from DEAE-cellulose in 0.001 M phosphate buffer. The purified human FABP is composed of two distinct protein bands which bind %PteGlu and resolves into 3 double bands on SDS polyacrylamide disc gel electrophoresis.

The purified goat FABP is a single protein band

which binds ^HPteGlu and remains a single band on SDS gel electrophoresis. Three peaks of FABP ranging from pH 6.6-8.4 are delineated by isoelectric

171 focusing of purified h u m a n and goat FABP.

The purified FABP's of human

and goat milk probably represent glycoproteins since they stain PAS positive and adhere to Concanavalin-A Sepharose affinity columns and elute w i t h alpha-methyl mannoside.

Thus, h u m a n FABP probably

represents

at least 3 proteins w h i c h include a poorly defined sedimentable m a c r o molecular weight protein and 2 basic glycoproteins w h i c h are dimers w i t h molecular weights of 30,000 w h i c h can form self-aggregates.

The

several peaks of folate binding activity obtained from purified FABP by isoelectric focusing may represent various folate binding proteins or perhaps the same protein w i t h conformational changes due to the p u r i fication process.

The latter explanation is probably true since pure

goat milk FABP appears to be a single protein immunologically and on SDS gel but gives several peaks of binding activity w i t h isoelectric focusing

(16).

PURIFICATION OF FOLATE Br:rr

Total Voiuiae Protein (r.l) (TIR)

Total FABP (¿Q Folic Acid "3ound)

PROÏÏÏIN i'KCK IfJKAN MILK Ret-cv^ry Oi Total FABP (Z)

Specific Activity (^g Folic Acid 3ound/n.q Protein)

Clarified Milk

6000

56,415

40.:

100

0.0007

Acid Charcoal Treated Milk

5000

7,350

38.7

95

0.005

5000

7,262

17.1

42

0.0025

Purification

Affm-ty Coiuirii LIuaLcs 1) li.iretamcd Xil.c

2) 0.02 Phosphate waiter it- .1 îi NaCI) 1000 pi: 7.2 1

3, 1 X iîâCl

500

0.16

0.3

0.18

0.4

15.2

37

4} 0.2 ìi Acetic Acid 50

22.5

25

1.2

1000

0.50

0.6

.vcn^ti.ration vita Sucrose Gradient Fraction (23.4%)

0.67

0.65 1.8

13.1

32

7.2

10,000

172 DEMONSTRATED EFFECTS OF FABP ON FOLATE PHYSIOLOGY Native FABP and purified FABP from several sources prevent the uptake of bound PteGlu and methylltyPteGlu into HeLa cell, Friend leukemia cell, L1210 murine leukemia and human lymphocyte cultures (25) (TABLE V). In contrast, FABP incubated with HeLa or L1210 leukemia cells enhance (60-100%) the uptake of subsequently added PteGlu at 5 minutes, which is recovered mainly in the membrane fraction (25). of PteGlu is not evident at 2 hours.

This enhanced uptake

This observation may relate to the

initial, rapid uptake of PteGlu in L1210 leukemia cells previously described (26).

The glycoprotein nature of FABP and the finding of

similar FABP in the lymphocyte membranes (24) and also in the brush border membranes of the rat small intestinal cell (18) suggest that FABP may be a component of a membrane carrier system for folic acid transport. TABLE V

THE EFFECT 01- l'lHT. HIHIAN MILK FABP ON THE UPTAKE OF % P t e G l u

Materials 3

HPteGlu Alone (Control)

3

HELA

CELTS

% Uptake of 0.5 ng HPteC]ii/107 Cells 7.2

3HPteG3vi and Pure FABP* Isoelectric Point pH 6.8

1.4

pH 7.5

1.1

pH 8.2

1.2

*FABP (7.4 iig PteGlu/bound/mg protein) recovered in the 3 isoelectric focusing points of human milk FABP. Ampholytes and sucrose were removed by G-25 Sephadex chromatography. Experiments were done in triplicate and were reproducible vithiri 107. error.

FABP appears to selectively bind a small amount of oxidized folates ( indicates inhibition. Abbreviations: F, folate; FH^, tetrahydrofolate; m 5 FH lt , 5-methyl tetrahydrofolate; MTX, Methotrexate. It is of interest that amethopterin is transported into cells via the second system; this result would not have been anticipated from superficial structural considerations.

Evidence supporting the existence of

this dual system has accumulated from several laboratories. findings (summarized in Table I) are as follows:

The relevant

182 Table I Folate Transport Systems in L1210 Cells

Compound Transported Property Inhibition by MTX

TI5FHL

MTX

Yes

Inhibition by mSFH^

No Yes

Inhibition by F

No

No

Inhibition by mercurials

Yes

Yes

Transport mutants (MTX-resistant)

Decreased

Decreased

No

No No change

(a) Competition experiments, carried out by Bertino and his colleagues [7], have shown that folate does not interfere with the uptake of either 5-methyl tetrahydrofolate or amethopterin (and vice versa), but the latter two compounds are mutually inhibitory,

(b) Mercurials block the transport

of 5-methyl tetrahydrofolate and amethopterin, but not folate [8,9].

(c)

Amethopterin-resistant mutants of LI210 c e l l s that owe their resistance to decreased transport of the drug are also defective with respect to uptake of 5-methyl tetrahydrofolate; their transport of folate, however, is normal [10]. Because the functional components have not yet been isolated from membranes, relatively l i t t l e i s known about the mechanism(s) by which folate compounds are brought into LI210 c e l l s by either of these two systems.

There

are some intriguing but unexplained data obtained with intact c e l l s :

(a)

Although metabolic energy i s required for transport, millimolar concentrations of glucose actually depress the uptake of 5-methyl tetrahydrofolate and amethopterin [11].

(b) Inhibitors such as iodoacetate [9,12], azide

[12], and v i n c r i s t i n e [13], cause increased uptake of amethopterin.

These

apparently unrelated agents may have in common the a b i l i t y to increase the

183 intracellular TPNH/TPN ratio.

Since TPNH is known to potentiate the bind-

ing of amethopterin to dihydrofolate reductase [14], this could account for an additional uptake of the drug.

As discovered by Hoffbrand et al. [15],

and confirmed in this laboratory [11], cyclic AMP inhibits the rate of uptake of folate compounds by LI210 cells.

In the latter experiments, two

devices were used to raise the level of cyclic nucleotides in the membrane; (a) addition of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine; and (b) addition of exogeneous cyclic nucleotides.

It is of

interest that dibutyryl cyclic GMP is considerably more effective than cyclic AMP, cyclic GMP or dibutyryl cyclic AMP [11].

B.

Bacterial Systems.

The advantages in using bacterial systems for studying transport processes lie in the fact that there is generally a greater uptake of metabolite per cell and that larger quantities of cells can be obtained for the isolation of components.

Lactobacillus

casei is ideally suited for this purpose

because of its high requirement for an external source of folate.

Trans-

port studies with these cells have followed the same general procedures used with mammalian cells.

The labeled compound is added to a suspension

of cells and uptake is monitored as a function of time.

As in the case

of mammalian cells, transport of folate compounds into L. casei is energyrequiring, concentrative and saturable [16-18].

In contrast, however, a

single system appears to be responsible for uptake of all folate compounds [17].

This is shown by several lines of evidence:

(a) Km values for the

transport of 5-methyl tetrahydrofolate and amethopterin are very similar to the Ki values obtained when these compounds are tested as inhibitors of folate uptake,

(b) Transport of various folate compounds is inhibited

to the same extent by increasing concentrations of iodoacetate.

(c)

Countertransport of 5-methyl tetrahydrofolate is enhanced by folate and amethopterin.

184-

Folate transport in L. aasei requires the presence of glucose.

The

apparent dependence upon glycolysis i s not surprising since this anaerobic organism lacks cytochrome and r e l i e s upon the fermentation of glucose to l a c t i c acid for energy production.

Further evidence bearing on this point

is provided by comparing the e f f e c t of inhibitors upon glycolysis and folate transport (Table I I ) .

There is a good parallelism between i n h i b i t i o n of

Table II Inhibitors of Folate Uptake and Glycolysis in Intact Cells of L. aasei

Per Cent of Control Compound

Cone. (mM)

Fluoride

20

68

69

100

5

7

0.5

44

138

5

13

185

0.5

87

78

5

2

3

1

78

80

5

4

5

85

91

2

4

Arsenate

Hydroxylamine

Iodoacetate

pCMS

0.2 5

Folate Uptake

Glycolysis

Folate transport (nmoles/1010 cells/10 min at 37°) and glycolysis (nmoles H+/1010 cells/10 min at 23°) were determined after preincubation of the c e l l s with the indicated i n h i b i t o r for 5 min at 37°. Folate, 1 yM; glucose, 10 mM.

185 the two processes for all compounds except arsenate, which inhibits folate transport but stimulates glycolysis.

The latter effect is apparently due

to the rapid arsenolysis of the acyl thioester intermediate generated during glyceraldehyde 3-phosphate oxidation and is similar to the uncoupling of oxidative phosphorylation by dinitrophenol. It is of interest that the transport of folate compounds into L. oasei cells is also inhibited by a variety of ionophores and sodium dodecyl sulfate (Fig. 2).

These compounds have little effect upon glycolysis and

IO

20

30

40

Inhibitor, /iM

Fig. 2. Inhibition of folate uptake in L. casei by ionophores and sodium dodecyl sulfate. Intact cells were preincubated for 5 min at 37° in the presence of the indicated concentrations of inhibitor prior to measurement of transport (10 min at 37°). Glucose, 10 mM; folate I yM. Valinomycin, (a gramicidin ( • • ) ; carbonylcyanide m-chlorophenylhydrazone (CCCP), ( a a ) ; pentachlorophenol (PCP), (• •); tetrachlorosalicylanilide (TCS), ( • •); and sodium dodecyl sulfate (SDS), (o o). thus appear to affect folate transport by a mechanism different from that of the glycolytic inhibitors.

Valinomycin and gramicidin (in contrast to

carbonylcyanide m-chlorophenylhydrazone, tetrachlorosalicylanilide, pentachlorophenol and sodium dodecyl sulfate) suppress folate transport at relatively low concentrations (< l yM); the lack of complete inhibition with these compounds may be referable to their limited solubility. bition by ionophores cannot be reversed by washing the cells.

Inhi-

186

When these ionophores are tested for their ability to cause a collapse of proton gradients across the cell membrane (Fig. 3), each of the ionophores which inhibits folate transport also enhances the rate of proton uptake by intact cells of L. case-L. However, the order of effectiveness of these compounds in facilitation of proton uptake is considerably different from their ability to inhibit folate transport.

These ionophores

seem to have a generally disruptive effect upon membranes, and thus folate transport does not appear to be linked directly to ion movement across the membranes.

5 50 •

Volinomycin + CCCP

2

3 4 5 Time, min

6

7

Fig. 3. Facilitation of proton uptake in L. easei by ionophores. Intact cells were washed with 50 mM potassium phosphate, pH 6.8, and suspended in 0.15 M KC1 (3.5 x 10 1 0 cells per ml). The pH was adjusted to 4.5 with 0.1 N HC1 and, after addition of the indicated ionophore, the timedependent increase in pH was recorded. Abbreviations as in Fig. 2.

Various metabolites have been examined for their ability to support folate transport (Table III).

Glucose, fructose, mannose and galactose

can be utilized by intact cells; glycolytic intermediates, ATP, DPNH and TPNH are ineffective.

However, when the cells are rendered more permeable

by treatment with lysozyme, several glycolytic intermediates can also support folate transport (Table III).

These latter compounds include

187 glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, and glyceraldehyde 3-phosphate.

It should be noted, however, that inter-

mediates beyond glyceraldehyde 3-phosphate in the glycolytic sequence (as well as ATP) cannot be used for this purpose.

Table III Energy Sources for Folate Uptake by Intact and Lysozyme-Treated Cells of L.

casei

Folate Uptake Energy Source

Intact Cells nmoles/10 1

0

Cells

Lysozyme-Treated Cells nmoles/10 mg protein

None

0. 4

0. 1

D-Glucose

3. 3

1..5

D-Fructose

3.,5

1. 5

D-Mannose

2..8

1.,6

D-Galactose

1..1

1.,1

D-Glucose 6-phosphate

0..4

3..4

D-Fructose 6-phosphate

0..3

3..4

D-Fructose 1,6-diphosphate

0..4

3,.2

DL-Glyceraldehyde 3-phosphate

0,.4

2 .8

1,3-Diphospho-D-glycerate

0 .4

0,.1

3-Phospho-D-glycerate

0 .4

0 .1

Phosphoenolpyruvate

0 .4

0 .1

Pyruvate

0 .4

0 .1

L-Lactate

0 .3

0 .1

Folate uptake by intact cells (in 0.05 M potassium phosphate, pH 6.8) or lysozyme-treated cells (in 0.05 M Tris-Cl, pH 7.4) was determined after incubation at 37° for 20 and 60 min, respectively. Folate, 1 yM; energy sources, 10 mM, except for 1,3-diphosphoglycerate, 4 mM.

188

Some additional insight into the mechanism of folate transport by L.

oasei

cells has been provided by a study of a mutant whose defective transport system can be repaired by the addition of mercaptoethanol

10

20

30

10 20 Time, min

30

10

20

(Fig. 4).

30

Fig. 4. Effect of 10 mM mercaptoethanol (ME) on the transport of folate compounds by mutant cells of L. oasei. Uptake at 37° of [ 3 H]folate, li+ [ C](dl ,L)5-methyl tetrahydrofolate and [ 3 H]amethopterin was determined as described previously [17].

Mercaptoethanol has little effect upon the uptake of folate by wild-type cells; when these cells are aged, however, some stimulation by mercaptoethanol is noted.

In the presence or absence of the thiol, the mutant

cells have approximately the same Km value as that of wild-type cells for the uptake of folate compounds (Table IV).

However, when binding of

folate to the mutant cells is examined (at 4° where transport is minimal), a biphasic curve is obtained (Fig. 5); the K^ values are 0.2 and 50 yM respectively, in the presence and absence of mercaptoethanol.

In contrast,

the K> t 3 N1

0

X

/No Addition

£ 25 1 § 20 S l alcohol-water-trichoroacetic

up to 22 ml in water)-29% ammonia

(225 CC 22 0 09)

could — The sodium salts

Table IV.

acid

(20 mg made

O

ä

229

3

O

1

'

•— I O M L I C I

600

Î J

D - erythro - H2 - Neopterm - PPP -

jj 500 0

1 100

_

• Enzyme

\

!

!

1 300

Uj

\

1

1

10 20 30 Fraction Number

A—

H- - Pterin - CH? OPP

K^

0

' D - erythro - H, - Neopterm - P

Formate

-Enzyme

X,

H,-Pterin

Pteroic Acid

- D- erythro - Neopteri

H, - Folic Acid

T~

Glutamats + 40

ATP

Fig. 9. Elution pattern from a Dowex-1 column of the radioactive product formed from H2-[a-^P]neopterin-PPP by the action of H2-neopterin-PPP pyrophosphohydrolase. See Suzuki and Brown (12) for details. Fig. 10. Enzymatic reactions necessary for the biosynthesis of h^-folic acid in E_. coli. Enzyme 1 is GTP cyclohydrolase I, enzyme 2 is H2neopterin-PPP pyrophosphohydrolase, enzyme 3 is a phosphatase, enzyme 4 is H2-neopterin aldolase, enzyme 5 is 6-hydroxymethyl-H2-pterin pyrophosphokinase, enzyme 6 is l^-pteroate synthetase, and enzyme 7 is ^-folate synthetase. results presented in Table IV show that the enzymatic product is inorganic pyrophosphate. The removal of inorganic pyrophosphate from the substrate should give, as the other product, D-erythro-H^-neopterin-P or the corresponding 2',3'cyclic phosphoester.

F^-Neopterin-cyclic-P has been reported to occur in

Methylococcus capsulatus (13).

We isolated the product of the action of

E. coli pyrophosphohydrolase by subjecting a reaction mixture to chromatography on Dowex-1 (CI ).

The column was developed by step-wise elution

with LiCl at concentrations of 0.2 M, 0.3 M and 1.0 M.

The data plotted

in Fig. 9 show that in the presence of the enzyme a product was formed which was eluted from the column with 0.3 M LiCl.

When no enzyme was in-

cluded, none of this material was produced; only the unreacted substrate I d e n t i f i c a t i o n o f the Product o f the A c t i o n o f H 2 -Neopterin-PPP Pyrophosphohydrolase as N e o p t e n n - P

Rp v a l u e s in S o l v e n t Compound Enzymatic product

Table V.

1

Neopterm

3

0.28

0.10

0.25

0.22

Enzymatic p r o d u c t , t r e a t e d w i t h phosphatase Neoptenn-cyclic-phosphate

2

4

0 77

0.28

0.28

SystenA

0.12 0.67 0.12

— See Suzuki and Brown (12) f o r composition o f these s o l v e n t

systems.

230 was evident.

The slight dip in the elution pattern of the unreacted sub-

strate resulted from a change in concentration of the LiCl from 0.3 M to 1.0 M.

The enzymatic product was concentrated and subjected to paper

chromatography as described by Suzuki and Brown (12).

The chromatographic

behavior of the product and the material generated from the product by treatment with alkaline phosphatase were compared with standards and the results are shown in Table V.

From these data, it is clear that the pro-

duct is not a cyclic phosphate and that treatment with alkaline phosphatase produces a compound with the chromatographic characteristics of neopterin. The product analyzed is the oxidized compound at this stage, rather than the dihydro form, because the pre-chromatographic treatment resulted in oxidation.

From all of the accumulated evidence, the conclusion can be

drawn that the enzymatic product is D-erythro-H^-neopterin-P. Since the phosphate esters of H^-neopterin cannot be used as substrate by dihydroneopterin aldolase (for the production of 6-hydroxymethyl-H2" pterin), an enzyme (or enzymes) must exist in E_. coli that will catalyze the hydrolytic removal of phosphate from D-erythro-H^-neopterin-P.

We have

determined that several such enzymatic activities are present in extracts of E_. coli, but we have not yet found one that is specific for this compound.

It is possible that this reaction does not depend on the action of

a specific phosphomonoesterase. With the discovery and study of these phosphatases, all of the seven enzymes in E_. coli necessary for the conversion of GTP, p-aminobenzoate and glutamic acid to dihydrofolate have been investigated in our laboratory. These enzymes and the reactions they catalyze

are shown in Fig. 10.

ENZYMATIC SYNTHESIS OF L-threo-H^,-NEOPTERIN Previous work from this laboratory (14) indicated that incubation of GTP with crude extracts of E_. coli resulted (after dephosphorylation) not only in the formation of D-erythro-HT-neopterin but also in the formation in approximately equal quantities of a threo-H,,-neopterin (whether D^ or _L was not established).

Confirmation that threo-neopterin occurs in E_. coli

was provided somewhat later by the isolation by Rembold and Heinz (15) of L-threo-neopterin from E_. coli•

The compound is also known to occur in

231 Comamonas sp. (16) where it is used as a cofactor for the oxygen-dependent hydroxylation of phenylalanine.

The work to be described below was direct-

ed toward an understanding of the enzymatic conversion (epimerization) of D-erythro-neopterin-PPP to a threo compound.

A detailed account of these

investigations will be published in the near future (17). Enzyme Assay Preliminary experimental results indicated that the substrate used for the production of the threo compound is D-erythro-H^-neopterin-PPP. enzyme would seem to be an epimerase.

Thus, the

Since the assay developed to mea-

sure epimerase activity depends on the separation of the dephosphorylated product from the dephosphorylated unreacted substrate, reaction mixtures were treated with alkaline phosphatase after incubation of the radioactive substrate with the epimerase.

This mixture was then subjected to paper

electrophoresis in the presence of a solution of 0.5% sodium borate to separate the radioactive threo-neopterin product from D-erythro-neopterin. The compounds analyzed on the ionopherograms are not in the dihydro forms since oxidation occurs during the procedure.

An analysis (by a strip

scanner) of an ionopherogram is presented in Fig. 11.

The results show

that threo-neopterin can be separated from erythro-neopterin by this method and that in the presence of the enzyme a radioactive product was formed that migrated as standard threo-neopterin.

This procedure does not allow

one to distinguish between L-threo-neopterin and D-threo-neopterin.

A

more detailed description of the assay can be found in another paper (17). For quantitative estimations of the formation of the product, the area of migration of the threo compound was cut from the paper and analyzed for radioactivity in a scintillation counter. Preparation of the Enzyme The epimerase used in the investigations to be reported below was prepared from a crude extract of E_. coli by steps involving the use successively of: fractionation with ammonium sulfate, chromatography on DEAE-Sephadex, chromatography on CM-Sephadex and filtration through Sephadex G-200. summary of this procedure is given in Table VI.

A

The increase in specific

232

- M

M-

O

threo origin

2

6

O

| erythro

8 16 16 20 22 Migration toward A n o d e . c m

Ö"

Guanosine 24

26

50 100 150 H z -Neopterin - P P P . ^ M

28

Fig. 11. Enzymatic formation of threo-neopterin from D-erythro-H2~neopterin-PPP and separation of erythro-neopterin from threo-neopterin by electrophoresis. Panel A gives results when incubation was carried out in the absence of epimerase and results in panel B were obtained from an incubation mixture to which epimerase was added. See Heine and Brown (17) for the details of this procedure. Fig. 12. Effect of substrate concentration on enzymatic epimerization of D-erythro-HT-neopterin-PPP to the threo compound. activity of the epimerase is only 7.5-fold; this results primarily because the enzyme is not very stable.

Although the purification achieved is not

very great, the fractionation procedure was worthwhile since it eliminated contaminating phosphatase activities so that an accurate assessment of the identity of the enzymatic product could be made.

Properties of the Epimerase The effect of substrate concentration on product formation is shown in Fig. 12.

Clearly, the rate of the reaction is dependent on substrate con-

centration, although no accurate measurement of the K

m

could be made be-

cause of the limitations of the accuracy of the assay, especially at low RELATIVE EFFECTIVENESS OF VARIOUS DIVALENT CATIONS AS STIMULATORS OF EPIMERASE ACTIVITY

S U M A R Y OF FRACTIONATION STEPS USED FOR THE PREPARATION OF THE EPIMERASE

Cation Enzyme Preparation

Specific Activity

Purification Factor

Overall Yield of Enzyme

Activity Relative to Mg per cent

units/mg

per cent

None

16

Crude extract

44

100

Mg2+

100

50-80% Amnonium sulfate fraction

27

Mn2+

106

28

Ca2'

84

Cu2*

24

Zn 2 *

13

Fe2+

8

DEAE-Sephadex fractions

106

CM-Sephadex fractions

252

Sephadex G-200 fractions

330

Table VI.

2 4 5 8 7.5

21 3.9 6.2

Table VII.

2+

253 substrate concentrations.

The reaction catalyzed by the epimerase is sti-

mulated significantly by the presence of a divalent cation. Table VII indicate that Mg^

+

and Mn^

+

slightly less so, and Cu^ , Zn^

+

+

The data of

are very effective, Ca^ + only

and Fe^ + have no significant effect.

The enzyme functions between pH 5.2 and 8.0 with a relatively broad optimum between pH 5.8 and 6.2. 50°.

Enzyme activity is maximal between 42° and

At temperatures higher than 50° the substrate is degraded nonenzyma-

tically to the cyclic monophosphate ester.

The activity of the enzyme is

stable to heating at 75° for one minute, but no activity remains after heating at 100° for one minute.

The enzyme can be stored for as long as

12 months at -20° with no loss of activity. The molecular weight of the epimerase has been estimated at 87,000-89,000 by filtration through a column of Sephadex G-200 calibrated with the standards: ribonuclease, chymotrypsin, ovalbumin, and rabbit muscle aldolase. Experiments designed to test the substrate specity of the epimerase have shown that D-erythro-H^-neopterin, D-erythro-H^-neopterin-P and D-erythroH2-neopterin-2',3'-cyclic-P are not substrates.

Other compounds that

would be reasonable to test (i.e., D-erythro-H^-neopterin-PP and L-erythroH^-neopterin-PPP) have not been available.

The fact that we have never

found that more than 50% of the substrate is converted to the product suggests that the reaction is reversible and that the equilibrium constant is close to unity.

However, we have never had enough of the product avail-

able to confirm this possibility.

Product In the routine assay used to determine enzymatic activity it is necessary to dephosphorylate the unreacted substrate and the product before analysis with electrophoresis.

We have attempted to devise methods to allow sepa-

ration of the product from the substrate without prior dephosphorylation but these have not been successful.

Thus, the routine assay gives no in-

formation about the number of phosphate groups retained in the product. Evidence bearing on this point was obtained with the use as substrate of 32 14 D-erythro-H 0 -[y- P,U- C]neopterin-PPP followed by an analysis of the

23^ P-containing products.

The results obtained from this experiment showed 14 that epimerization had occurred (i.e., C-containing threo-neopterin was identified by the use of the routine assay procedure), but that no radioactive orthophosphate or pyrophosphate was produced by the action of the epimerase.

Thus, the product is undoubtedly a triphospho ester.

The product analyzed in the routine assay was always a pterin although the substrate was a dihydropterin.

During the preparation of the samples for

electrophoresis, one of the procedures is to evaporate the material to dryness.

We have determined that during this process dihydropterins be-

come oxidized to pterins.

The evidence seems clear, however, that the

product of the action of the epimerase is a dihydropterin (i.e., no oxidation occurred) since we observed no decrease in the absorba :e peak at 330 nm (typical for 7,8-dihydropterins) during the enzymatic reaction. The electrophoretic method used in the assay will separate erythro-neopterin and threo-neopterin, but it will not distinguish D-threo-neopterin from L-threo-neopterin.

In order to decide whether the product is the £

or L stereoisomer, we devised a method based on the fact that D-erythrot^-neopterin and L-threo-H^-neopterin are used equally well as substrate for dihydroneopterin aldolase, whereas I^-threo-h^-neopterin is not used at all (11).

In this experiment, after the action of the epimerase and

treatment with phosphatase (to dephosphorylate completely the product and the unreacted substrate), aldolase was added to the reaction mixture along with other components needed for the conversion of the product of the action of the aldolase (6-hydroxymethyl-H.-pterin) to H -pteroic acid. 14 2+ These components include ATP, [ C]p-aminobenzoate, Mg and the enzymes hydroxymethyl-H^-pterin pyrophosphokinase and H^-pteroic acid synthetase. After incubation with these materials, the amount of l^-pteroic acid formed was determined.

Another series of reaction mixtures was prepared

to contain graded amounts of D-erythro-H^-neopterin-PPP, but no epimerase, and the amounts of t^-pteroic acid produced as a function of the concentration of this substrate were determined.

From the regular assay for

epimerization it was determined that of the 0.92 nmoles of D-erythro-H,,neopterin-PPP added as substrate, 0.33 nmoles were epimerized.

If the

threo product had been D-threo, only the unreacted substrate, equal to

235

Fig. 13. Identification of the product of the action of the epimerase as the L-threo stereoisomer. The preparation of the standard curve (shown in the figure) for the conversion of D-erythro-H^-neopterin-PPP to H 2 pteroic acid in the presence of alkaline phosphatase, dihydroneopterin aldolase, hydroxymethyl-H2-pterln pyrophosphokinase, H2-pteroic acid synthetase, [^CJp-AB, ATP and M g 2 + is described briefly in the text and more completely elsewhere (17). The arrow in the figure shows the amount of ^ - p t e r o i c acid synthesized from materials that were present in a reaction mixture in which D-erythro-l^-neopterin-PPP had been incubated with epimerase prior to incubation with the components used to prepare the standard curve. Fig. 14.

Reaction catalyzed by D-erythro-H^-neopterin-PPP

2'-epimerase.

0.59 nmoles of D-erythro-neopterin, would have been available as substrate for the aldolase.

From the standard curve shown in Fig. 13, 0.59

nmoles should have allowed the production of pteroic acid equivalent to 6400 cpm; however, the analysis showed that the amount of pteroic acid synthesized from material that had been subjected to the action of the epimerase was equal to 9800 cpm, an amount that would have been produced from 0.92 nmoles of the D-erythro-H^-neopterin-PPP offered as substrate (see standard curve, Fig. 13).

Thus, the threo product could not have

been the D-threo stereoisomer.

From these results we conclude that the

reaction catalyzed by the epimerase is the conversion of D-erythro-H^neopterin-PPP to L-threo-H^-neopterin-PPP as shown in Fig. 14, and we have therefore chosen to call the enzyme "D-erythro-H^-neopterin-PPP 2'-epimerase".

236 Biochemical Significance of L-threo-Neopterin in

coli

Although it is clear from our results and those of Rembold and Heinz (15) that L-threo-neopterin (or the reduced form) occurs in E_. coli, the biochemical function of this compound in E_. coli remains unknown.

This sub-

stance is needed for the oxygen-dependent hydroxylation of phenylalanine in Comamonas sp. (16), but such a reaction is not known to occur in E_. coli. Another question that arises is why biosynthetic L-threo-H^-neopterin accumulates in E_. coli since it is known to be used as substrate by dihydroneopterin aldolase (11).

One possibility that should be considered

is that a phosphorylated form of L-threo-H,,-neopterin is the functional form in E_. coli.

This would explain why it would not be used for the bio-

synthesis of H 2 -folic acid since the phosphorylated compounds are not substrates for the aldolase (11).

In this connection it should be mentioned

that neopterin monophosphate was isolated from E_. coli by Goto and Forrest (18), although it was not established whether the compound was threo or erythro.

GTP CYCLOHYDROLASE II A second GTP cyclohydrolase in E_. coli was discovered in our laboratory (19) as a result of an attempt to modify the previously published fractionation procedure for GTP cyclohydrolase I (10).

We have chosen to call

this second enzyme GTP cyclohydrolase II to distinguish it from the one first investigated.

One of the modifications introduced in the fraction-

ation scheme was to subject a dialyzed extract of IE. coli to chromatography on a column of DEAE-Seaphdex A-50. buffer lacking EDTA.

The column was developed with

EDTA had always been

included previously because

it stabilizes the activity of GTP cyclohydrolase I (10).

The pattern of

elution of activity that converts carbon 8 of GTP to formate is shown in Fig. 15.

Two peaks of activity are evident.

Material in the first peak

(fractions 48-58) resembled the GTP cyclohydrolase described by Burg and Brown (10) in its resistance to inhibition by EDTA.

The enzyme activity

in the second peak (fractions 65-75) is stimulated by Mg^ + and is completely inhibited by EDTA.

237 *—X

A 280

o

l 4

c

Released

•

S

P

Released

2.0

i.5

1.0

0.5

0

10

2 0

30

4 0

Fraction

5 0

S O

70

80

90

30

Number

50

70

SO

Fraction Number

Fig. 15. Chromatography on DEAE-Sephadex of a dialyzed extract of E_. coli. GTP cyclohydrolase activity of the fractions from the column was determined and the column was operated as described in another paper (19). Fig. 16. Fractionation of GTP cyclohydrolase II on Sephadex G-200. Foor and Brown (19) for details.

See

Purification of GTP Cyclohydrolase II A crude extract of

coli was made by treating the cells with lysozyme.

The resulting mixture was treated with DNase and subjected to centrifugation to remove insoluble material.

The resulting extract was subjected

to fractionation with ammonium sulfate, chromatography on DEAE-Sephadex, filtration through Sephadex G-200 and chromatography on a hydroxylapatite column to yield a preparation whose specific activity was 2200 times as great as that of the crude extract. is given in Table VIII.

A summary of the purification scheme

The elution pattern of the enzyme from a Sephadex

G-200 column is shown in Fig. 16.

In the figure are plotted enzyme acti-

vities for removal of carbon 8 of GTP as formate (measured with the use as substrate of [8- 14 C]GTP) and for removal of

32

P from [y- 3 2 P]GTP.

data show that the two activities elute together.

The

This suggests that

the same enzyme is responsible for both activities.

In the final purifi-

cation step (chromatography on hydroxylapatite) the two activities also 32 elute together and again P release and formate production were stoichiometric .

SUMMARY OF PURIFICATION OF GTP CVCLOUYDROLASr

Preparation

Table VIII.

Total Activity

Specific Activity

units

munits/mg protein

Crude extract

3 5

0 10

Ammonium sulfate fraction

3 4

0 18

DEAE-Sephadex eluate

2 8

8 1

Sephadex G-200 eluate

2 7

69 0

Hydroxylapatite eluate

223 0

e o a

238 Properties of GTP Cyclohydrolase II GTP cyclohydrolase II differs dramatically from GTP cyclohydrolase I in that M g 2 + is required for activity of enzyme II.

Of a group of divalent

ions tested (Ba 2 + , C a 2 + , C o 2 + , C u 2 + , F e 2 + , M g 2 + , M n 2 + and Z n 2 + ) only M g 2 + and M n 2 + were effective and M n 2 + was only 70% as effective as M g 2 + tested at 0.25 mM).

(both

Maximal stimulation was achieved with 150 pM M g 2 + ;

half-maximal at 14 pM. The enzyme functions at pH values of 6.5 and higher with an optimum at pH 8.4.

The molecular weight of the enzyme was estimated at 44,000 from

its position of elution from a calibrated column of Sephadex G-200.

The

apparent molecular weight was independent of ionic strength. The initial velocity of the reaction catalyzed by the enzyme is proportional to the enzyme concentration, but the velocity decreases steadily during the course of the reaction, probably because of the formation of inorganic pyrophosphate, one of the products of the reaction which is also an inhibitor (see below).

The K m for GTP was estimated as 41 pM from an

Eadie and Hofstee plot. The enzyme is quite specific for GTP as substrate.

Compounds found not to

be substrates are: dGTP, GDP, GMP, guanosine, guanine, 3-Y-methylene GTP, ATP, ITP and XTP.

Guanosine-5 1 -tetraphosphate was used as substrate,

but only about a third as well as GTP. the reaction is inorganic pyrophosphate.

The most effective inhibitor of At a concentration of 0.2 mM,

this substance inhibited both the production of formate and inorganic pyrophosphate by 52%.

Products Preliminary information was obtained on the nature of the products formed from GTP by following the change in the ultraviolet absorption spectrum during the course of the reaction.

It is clear that a F^-neopterin com-

pound is not formed during the reaction since no absorption band at 330 nm (characteristic of t^-neopterin) was evident. appeared during the incubation.

Instead a peak at 287 nm

This peak disappeared at lengthened

239 incubation times as the release of formate from GTP approached completion. We found that the peak at 287 nm could be preserved by including 7 mM mercaptoethanol in the reaction mixture.

The appearance of the absorption

band at 287 nm suggested that a pyrimidine is formed as a product.

A

likely candidate is 6-hydroxy-2,4,5-triaminopyrimidine or the corresponding compound with a phosphorylated ribose attached to the 4-amino group. Synthetic 6-hydroxy-2,4,5-triaminopyrimidine was examined and found to resemble the enzymatic product in that the two exhibit similar absorption spectra and the absorption peak at 290 nm of the synthetic compound also decays with incubation at 37°.

This could be prevented by mercaptoethanol.

The spectral changes accompanying the enzymatic reaction are shown in Fig. 17. 14 With [U-

C]GTP as substrate the only radioactive product not adsorbable

to charcoal is formate when the charcoal treatment is carried out immediately after incubation.

However, when incubated reaction mixtures were

not treated with charcoal until 24 hours after the enzymatic reaction, much more radioactivity (equal to approximately 40% of the total) was not found capable of being adsorbed to charcoal.

We suspected that either

ribose or ribose-P had been released during this period.

We tested this

hypothesis by treating the material with phosphatase and analyzing for ribose by paper chromatography.

The radioactive material migrated as

Fig. 17. Ultraviolet absorption spectrum of a reaction mixture ing GTP and GTP cyclohydrolase II) at various incubation times. bers on the figure refer to incubation time in minutes.

(containThe num-

Fig. 18. The reaction catalyzed by GTP cyclohydrolase II and the conversion of the resulting product to pterin by the nonenzymatic reaction with glyoxal.

24-0

ribose in two separate chromatographic solvent systems.

This suggested

that ribose was not removed enzymatically, but was released over the storage period because of the instability of the enzymatic product. To obtain further information about the nature of the products, the enzyme was incubated with [U-14C]GTP along with either [a-32P]GTP or [y-32P]GTP. The incubated mixtures were subjected to chromatography on DEAE-Sephadex A-25 to separate the products.

The details of this experiment along with

the resulting elution pattern from the column are presented in another publication (19).

Briefly, the results indicated that the following pro-

ducts could be separated and identified: formate, inorganic pyrophosphate, and a 9-carbon compound that also contained what was originally the (Diphosphate residue of the substrate (GTP). pyrophosphate.

The y-phosphate appeared in the 14 32 Table IX contains a summary of the amounts of C- and P-

labeled materials produced from the radioactive GTP.

Equimolar quantities

of formate, inorganic pyrophosphate and 9-carbon compound thought to be 2,S-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate were produced.

Such an orth-diaminopyrimidine should react with dicarbonyl com-

pounds to yield pteridines.

We were successful in devising conditions

under which the radioactive enzymatic product would react with glyoxal to yield ribose-5-P and 2-amino-4-hydroxypteridine (pterin).

The latter

compound was identified by comparison of its paper chromatographic properties with standard pterin.

The product was also shown to be identical

with standard pterin in its fluorescence emission spectrum. All of the accumulated evidence indicates that the enzymatic and nonenzymatic reactions described above proceed as shown in Fig. 18 to produce (enzymatically) a ribose-5-P derivative of hydroxytriaminopyrimidine which AMOUNTS OF PRODUCTS FORMED FROM GTP BY INCUBATION WITH GTP CYCLOHYDROLASE II Amount Produced, Based on Radioactivity from Substrate [U- 1 4 C,a- 3 2 P]GTP

Table IX.

Compound Produced Enzymatically

14

From C content

From 3 2 P content

From C content

From 3 2 P content

nmoles

nmoles

nmoles

nmoles

Formate

11.0

C 9 -Product

10.9

PP

i

[U- 1 4 C,Y- 3 2 PJGTP

11.0 11.5 0

11.0

0 11.4

24-1

is then converted (nonenzymatically) to pterin and ribose-5-P in the presence of glyoxal.

It is not surprising that the enzymatic product shown

in Fig. 18 is so unstable, since Plaut (20) has shown that a similar compound (5-amino-2,6-dihydroxy-4- (1'-I)-ribitylamino)pyrimidine) , which is produced during the biosynthesis of riboflavin, is degraded in the presence of C>2 to ribitylamine and alloxan.

By analogy, the enzymatic pro-

duct shown in Fig. 18 would be degraded to an orthoquinone compound followed by hydrolysis to yield 5-phosphoribosylamine, an unstable compound that is easily hydrolyzed to ribose-5-phosphate and ammonia.

The identi-

fication of ribose (after treatment with phosphatase) as a degradation product of the enzymatic product is consistent with such a reaction scheme. Significance of the Presence of GTP Cyclohydrolase II in E. coli The evidence suggests that GTP cyclohydrolase II catalyzes both formate release (from carbon 8 of GTP) and pyrophosphate formation.

These two

products along with the pyrimidine product are all formed in stoichiometric amounts.

The alternative possibility that two enzymes are involved cannot

be ruled out entirely, but it does seem unlikely since: (a) catalytic activities for formate and pyrophosphate formation purify together as if a single protein is involved; (b) GMP cannot be a free intermediate because it cannot be used as substrate for formate formation; (c) it is unlikely that a triphosphate derivative produced by removal of carbon 8 from GTP is a free intermediate because kinetic studies indicate that the rates of formate and pyrophosphate formation from GTP are identical; and (d) we have determined that pyrophosphate inhibits the enzymatic production of formate and pyrophosphate to the same extent. Another question that arises is the possible relation of GTP cyclohydrolases land II. two

are

The accumulated evidence conclusively indicate that the

completely different enzymes.

This is based on the following

facts: (a) the products are different; (b) enzyme II needs Mg^ + whereas enzyme I does not; (c) the K^ values for GTP of the two enzymes are vastly different; (d) the molecular weights are different; and (e) enzyme I is heat stable and enzyme II is not. A consideration of the biochemical significance of GTP cyclohydrolase II

242 in E^ coli leads to speculation about its possible role in the biosynthesis of riboflavin.

Other workers have clearly shown that a guanine com-

pound is the precursor of riboflavin (21-23),

and

riboflavin-requiring

mutants of yeast are known to excrete a metabolite that reacts nonenzymatically with 2,3-butanedione to give 6,7-dimethylpterin (24).

This meta-

bolite is thought to be 6-hydroxy-2,4,5-triaminopyrimidine or perhaps a ribosylated derivative of this pyrimidine.

Our results indicate a 5-

phosphoribosylated derivative of this pyrimidine is formed as a product of the action of GTP cyclohydrolase II.

If this compound is an interme-

diate in riboflavin biosynthesis, we would expect it to be metabolized further by enzymes that successively: (a) either reduce the ribose group to ribityl or replace the ribose with ribityl; (b) convert the 2-amino group to a hydroxy group; and finally add an unidentified 4-carbon compound to yield 6,7-dimethyl-Y-ribityllumazine, a recognized precursor of riboflavin (21).

Acknowledgements. The authors are indebted to Mrs. Elaine Lenk of the Biology Department at the Massachusetts Institute of Technology for the electron micrograph. The experimental work reported in this paper was supported by research grants from the National Institutes of Health of the U.S. Public Health Service (grant number AM03442) and the National Science Foundation (grant number GB33929X1). REFERENCES 1. Brown, G.M.: Enzymic synthesis of pterins and dihydropteroic acid. Chemistry and Biology of Pteridines (ed. by Iwai, K., Akino, M., Goto, M., and Iwanami, Y.), Int'l. Academic Printing Co., Ltd., Tokyo, 1970, pp. 243-264. 2. Shiota, T and Palumbo, M.P.: Enzymatic synthesis of the pteridine moiety of dihydrofolate from guanine nucleotides. J. Biol. Chem. 240, 4449-4453 (1965). 3. Guroff, G. and Strenkoski, C.A.: Biosynthesis of pteridines and of phenylalanine hydroxylase cofactor in cell-free extracts of Pseudomonas species (ATCC 11299a). J. Biol. Chem. 241, 2220-2227 (1966). 4. Levenberg, B. and Kaczmarek, D.K.: Enzymic release of carbon 8 from guanosine triphosphate, an early reaction in the conversion of purines to pteridines. Biochim. Biophys. Acta 117, 272-275 (1966). 5. Mitsuda, H., Suzuki, Y., Tadera, K. and Kawai, F.: Biochemical studies on pteridines in plants II. Biogenesis of folic acid in green leaves: enzymatic synthesis of dihydropteroic acid from guanosine compounds and mechanism of its synthetic pathway.J. Vitaminol. 12, 192-204 (1966).

24-3

6. Brown, G.M. and Fan, C.L.: Synthesis of pteridines catalyzed by enzymes from Drosophila melanogaster. This Symposium, pp. 7. Jackson, R.J., Wolcott, R.M. and Shiota, T.: The preparation of a modified GTP-Sepharose derivative and its use in the purification of dihydroneopterin triphosphate synthetase, the first enzyme in folate biosynthesis. Biochem. Biophys. Res. Communs. 51, 428-435 (1973). 8. Davis, B.J.: Disc electrophoresis II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427 (1964). 9. Shiota, T., Jackson, R. and Baugh, C.M.: Biosynthetic pathway of dihydrofolate. Chemistry and Biology of Pteridines (ed. Iwai, K., Akino, M., Goto, M. and Iwanami, Y.), Int'l. Academic Priting Co., Ltd., Tokyo, 1970, pp. 265-280. 10. Burg, A.W. and Brown, G.M.: The biosynthesis of folic acid VIII. Purification and properties of the enzyme that catalyzes the production of formate from carbon 8 of guanosine triphosphate. J. Biol. Chem. 243, 2349-2358 (1968). 11. Mathis, J.B. and Brown, G.M.: The biosynthesis of folic acid XI. Purification and properties of dihydroneopterin aldolase. J. Biol. Chem. 245, 3015-3025 (1970). 12. Suzuki, Y. and Brown, G.M.: The biosynthesis of folic acid XII. Purification and properties of dihydroneopterin triphosphate pyrophosphohydrolase. J. Biol. Chem. 249, 2405-2410 (1974). 13. Urushibara, T., Forrest, H.S., Hoare, D.S. and Patel, R.N.: Pteridines produced by Methylococcus capsulatus. Isolation and identification of a neopterin 2':3'-phosphate. Biochem. J. 125, 141-146 (1971). 14. Jones, T.H.D. and Brown, G.M.: The biosynthesis of folic acid VII. Enzymatic synthesis of pteridines from guanosine triphosphate. J. Biol. Chem. 242, 3989-3997 (1967). 15. Rembold, H. and Heinz, G.: L-threo-neopterin, the major pterin in Escherichia coli B. Hoppe-Seyler's Z. Physiol. Chem. 342, 1271-1272 (1971). 16. Guroff, G. and Rhoads, C.A.: Phenylalanine hydroxylation by Pseudomonas species (ATCC 11299a). J. Biol. Chem. 244, 142-146 (1969). 17. Heine, M.C. and Brown, G.M.: Enzymatic epimerization of D-erythro-dihydroneopterin triphosphate to L-threo-dihydroneopterin trTphosphate. In — preparation. 18. Goto, M. and Forrest, H.S.: Identification of a new phosphorylated pteridine from E^ coli. Biochem. Biophys. Res. Communs. 180-183 (1961). 19. Foor, F. and Brown, G.M.: Purification and properties of GTP cyclohydrolase II from Escherichia coli. J. Biol. Chem., in press (1975). 20. Plaut, G.W.E.: Studies on the nature of the enzymic conversion of 6,7dimethyl-8-ribityllumazine to riboflavin. J. Biol. Chem. 238, 22252243 (1963).

24-4

21. Plaut, G.W.E.: The biosynthesis of riboflavin. Comprehensive Biochemistry (Vol. 21, ed. Florkin, M. and Stotz, E.), Elsevier Publishing Co., New York, 1971, pp. 11-45. 22. Bacher, A. and Lingens, F.: The structure of the purine precursor in riboflavin synthesis. Angew. Chem. 81_, 393-394 (1969); Angew. Chem. , Int'l. Edition English 8_, 371-372 (1969). 23. Baugh, C.M. and Krumdieck, C.L.: Biosynthesis of riboflavine in Corynebacterium species: the purine precursor. J. Bacterid. 98, 1114-1119 (1969). 24. Bacher, A. and Lingens, F.: Biosynthesis of riboflavin. Formation of 6-hydroxy-2,4,5-triaminopyrimidine in Riby mutants of Saccharomyces cerevisiae. J. Biol. Chem. 246, 7018-7022 (1971). DISCUSSION Elstner: You call your enzyme no. II "Cyclohydrolase II" - but this enzyme does not cyclize; - would'nt be the correct name for this enzyme; GTP-8formyl hydrolase as proposed for an enzyme from Streptomyces ? G.M.Brown: You have a point - we refer to the enzyme as GTP-cyclohydrolase merely for convenience. Wood: Use of a-ketoesters or a-keto acids in place of a-dicarbonyl compounds (such as glyoxal) in characterization of Cg compound might be useful. This should lead to 7-oxopteridine-glycosides. G.M.Brown: Thank you for the suggestion. Wagner: Is there any evidence for carbohydrate being associated with cyclohydrolase I ? G.M.Brown: None, but we probably have not looked carefully enough to eliminate this possibility. Plaut: Why do you insist that all intermediates on the way to riboflavin have to be phosphorylated - i.e., assuming your cyclohydrolase II is on the path ? G.M.Brown: I do not eliminate any possibilities. Plaut: I want to point out that while riboflavin binds to riboflavin synthetase - EMN does not. G.M.Brown: That does appear to eliminate riboflavin phosphate as a final product. Buff: The question is regarding to the widely different Km values of your enzyme. What is the concentration of GTP in the E.coli cell ? G.M.Brown: We are, of course, interested in this. We have not yet received an answer from any of my microbiologist friends.

24-5

Albert: Is there anything in your new work which would throw light on the biosynthesis of biopterin ? G.M.Brown:

I may be able to throw some light on this in my next paper.

Scrimgeour: Your GTP cyclohydrolase I seems to break the golden rule" (or tin rule ?) that oligomeric proteins are extremely unstable, especially toward heat, Is it cold labile ? G.M.Brown:

It is not cold labile.

Wrigglesworth: Is the GTP cyclohydrolase II found in mammalian systems ? G.M.Brown:

I don't know.

Lund: Do you have any proposal for the reaction mechanism for the epimerization of D-erythro-dihydroneopterin PPP ? G.M.Brown:

We have not had enough of the enzyme to do such studies.

Lund: Would it be a reasonable suggestion that the azomethine group in the dihydropteridine nucleus plays the same role in the epimerization reaction as the carbonyl function does in the epimerization reaction of carbohydrates ? G.M.Brown: It would be reasonable if the epimerization were on the l^-carbon. Unfortunately, the reaction occurs on the 2-carbon and thus the azomethine group would not be as important in the activation process. Montgomery: I suggest using nitrous acid to trap the intermediate pyrimid m e from the cyclodehydrase II :

HO OH

HO OH

In this way the ribose phosphate moiety will not be lost and the exact nature of the pyrimidine will be established. 8-Azaguanylic acid has been synthesized and well characterized. G.M.Brown:

Thank you. That seems to be a good idea.

Biosynthesis of Pterins in Mammalian Systems K. Fukushima, I. Eto, T. Mayumi, W. Richter, S. Goodson and T. Shiota

The questions regarding the origin and the biosynthesis of biopterin has been of great interest to biologists for a number of years, and much effort has been directed toward the elucidation of answers to these questions. The pathway for the synthesis of pterins and of folate in Escherichia coli (1), Lactobacillus plantarum (2), Commamonas (3), Serratia indica (4) and Tetrahymena (5) as well as information on the biosynthesis of biopterin in bullfrog tadpoles (6-8) and f r u i t f l i e s (9) has been reported.

In mamma-

lian systems, the possible synthesis of biopterin has been suggested (1012), however, definitive evidence of the o r i g i n and for biopterin synthesis was t o t a l l y lacking until very recently.

We reported that in growing c u l -

tures of Chinese hamster ovary c e l l s (13), f o l i c acid i s not a precursor of biopterin but i s a precursor of pterin, 6-hydroxymethylpterin and 6carboxypterin.

Moreover, from these r e s u l t s , the hypothesis for the addi-

tion of a three carbon unit to pterin or a two carbon unit to 6-hydroxymethylpterin i s not supported.

Additional studies using [2-^C]guanine,

[8-^C]guanine, [U-^C]guanosine or [6-^C]glucose as a precursor of biopterin indicated that in the Chinese hamster ovary cell cultures, guanosine or i t s nucleotide i s converted to biopterin and that reactions such as

ring-opening, elimination of carbon atom 8 and ring closure by carbon

atoms 1' and 2' of ribose are involved.

A subsequent report by Buff and

Dairman (14), who worked with two neuroblastoma cell l i n e s , also concluded that a guanosine-1ike compound was the precursor for biopterin.

24-8

In this presentation, results of recent work will be presented demonstrating the origin and the synthesis of biopterin in mammalian systems. Material and Methods Primary cultures - Primary cultures of various organs were prepared from rats (Fisher), mice (3CH/HeJ), Chinese hamsters and Syrian golden hamsters employing usual cell culture techniques.

The cells were grown in F-12 (15)

medium containing 10% fetal calf serum with the medium being changed every other day.

The cultures were grown in a 5% C02-air incubator.

When the

cells became approximately 10% confluent, the medium was removed and the attached cells washed several times with F-12 medium supplemented with 10% charcoal-treated fetal calf serum and incubated in this medium without further changing until the cells were confluent.

The supernatant

fluid was then removed and assayed for Crithidia activity. Homogenate preparations - From (adult) Syrian golden hamsters, various organs were homogenized in either 0.01 M Tris-0.04 M KC1 buffer, pH 8.0 or in 0.05 M potassium phosphate buffer, pH 6.8 to give a 20-25 percent (wet weight per volume) preparation.

In certain of the experiments either the

crude homogenate preparations or dialyzed supernatant fractions of these crude preparations were used.

In other experiments, however, a Sephadex

G-150 fraction (Fig. 1) or an Ultragel AcA-34 (Fig. 2) fraction was used. To obtain these preparations, supernatant fluids from crude liver preparations were concentrated against solid sucrose, dialyzed against 0.005 M Tris-HCl buffer, pH 8.0 and applied to either a Sephadex G-150 column or an Ultragel AcA 34 column and the column washed with the same buffer.

The

fractions which were collected were measured for protein absorbance at 280 nm and also assayed for the production of fluorescent materials by incuba-

24-9

ting with GTP for three hours at 37°C.

Fractions producing peak fluore-

scence were pooled and used in certain experiments.

Fig. 1. Sephadex G-150 column chromatography of a supernatant fluid of a liver homogenate. A 2 ml portion (220 mg of protein) of a concentrated supernatant fluid of a liver homogenate was applied to a Sephadex G-150 column. Fig. 2. Ultragel AcA-34 column chromatography of a supernatant fluid of a liver homogenate. A 10 ml portion (1.1 g of protein) of a concentrated supernatant fluid of a liver homogenate was applied to an Ultragel AcA-34 column. Assay system for the utilization of guanine derivatives - In a total volume of 0.15 ml, the reaction mixture contained:

GTP or other purine de-

rivatives, 0.3 ymoles; Tris-HCl , pH 8.0, 75 ymoles and a homogenate preparation.

In some cases, 0.15 ymole each of NADH, NADPH, NAD and NADP was

included.

The mixture was incubated at 37° in the dark for 3 hours at

which time 2 ml of 0.1 N HC1 were added to terminate the reaction.

For

purposes of oxidizing any reduced pterins, the reaction mixture was treated with iodine.

The procedure consisted of adding one drop of 1% starch

solution and 0.01 ml of iodine solution (2 g of KI and 1 g of iodine in 100 ml of water) to the terminated reaction mixture.

After 15 minutes,

ascorbic acid solution was added to reduce the excess iodine and then 0.2 ml of 2 M Tris base was added to adjust the pH of the mixture to 8.0.

The

reaction mixture was either directly assayed for the production of Crithidia active substances or processed by a chromatography procedure to

250 isolate pterins. Assay system for the u t i l i z a t i o n of D-erythrodih.ydroneopterin triphosphate Either a mixture or individual crude homogenates of brain, lung, l i v e r and kidney were prepared from the adult Syrian golden hamster in 0.05 M potassium phosphate buffer, pH 6.8.

For the preparation of dialyzed superna-

tant fractions, crude homogenates were centrifuged and the supernatant f l u i d s dialyzed against the same buffer. A two-stage experiment was performed to test for the u t i l i z a t i o n of Derythrodihydroneopterin triphosphate by crude homogenates or dialyzed supernatant fractions of hamster organs. contain:

The f i r s t stage was prepared to

Tris-HCl, pH 8.0 12 ymoles; KC1 , 60 ymoles; D-er.ythrodihydro-

neopterin triphosphate synthetase preparation from L_. plantarum (16) (specific a c t i v i t y :

7.2 units/mg protein) equivalent to 5 mg of protein;

and [U- 14 C]GTP (1 y C i ) , 0.6 ymole in a total volume of 0.3 ml and the mixture incubated for 2 hours at 37° in the dark.

At 2 hours (second stage),

the following materials were added to the f i r s t stage mixtures to give a total volume of 1.0 ml:

potassium phosphate buffer, pH 6.8, 50 ymoles;

MgS04, ° - 8 umole; NAD, 10 ymoles; NADP, 5 ymoles; NADH and NADPH, 2.5 ymoles each and either the crude homogenate or the dialyzed supernatant fraction.

This reaction mixture was incubated for 5 hours at 37° in the

dark, at which time the reaction was terminated by the addition of 0.2 ml of 1 N HC1, and the pterins oxidized by iodine as described previously. The reaction mixture was assayed for the production of Crithidia active substances or processed by a chromatography procedure to purify and to isolate the pterins produced. Chromatography procedure - The iodine-treatment reaction mixture was in

251 most cases treated with a l k a l i n e phosphatase and the pH of the s o l u t i o n adjusted to 1.0.

The s o l u t i o n was added to a Dowex-50 (H + ) column, washed

with water and eluted with 1 N NH4OH.

The eluate was concentrated and ap-

p l i e d to an ECTEOLA-Sephadex (OH") column which was washed with water and eluted with 0.5 M a c e t i c a c i d .

The eluate was concentrated and added to an

ECTEOLA-Sephadex pH 7 column which was eluted with water.

The f i n a l

eluted

material was concentrated and applied to c e l l u l o s e TLC sheets and the sheets developed in the appropriate s o l v e n t systems depending upon the experiment.

The s o l v e n t s are:

ethylacetate-water

Solvent A, 3% NfyCl ; Solvent B, 1 - p r o p a n o l -

( 7 : 1 : 2 ) ; Solvent C, ethanol-ammonium borate (pH 8.0 as

5% boric a c i d ) - 3 % N H 4 C I

( 2 : 1 : 1 ) ; Solvent D, l - p r o p a n o l - 1 % NH4OH

(2:1);

Solvent E, 1 - b u t a n o l - a c e t i c acid-water ( 4 : 1 : 2 ) ; Solvent F, acetone-ammonium borate (pH 8.2 as 5% boric acid)-3% N H 4 C I

(1:1:1) and Solvent G, e t h a n o l -

ammonium borate (pH 8.2 as 5% boric acid)-3% N H 4 C I

(2:1:1).

For the quan-

t i t a t i v e r a d i o a c t i v e a s s a y s , c a r r i e r b i o p t e r i n and D-erythroneopterin were spotted with the column p u r i f i e d material developed in Solvent B.

to TLC sheets and the sheets

The spots corresponding to b i o p t e r i n and D - e r y t h r o -

neopterin were removed, eluted with water, concentrated and reapplied to TLC sheets and the sheets developed in Solvent /t.

F i n a l l y the spots c o r -

responding to authentic b i o p t e r i n and D-erythroneopterin were removed and assayed f o r

radioactivity. Results

Production of C r i t h i d i a a c t i v e substances by primary t i s s u e c u l t u r e s - The production of C r i t h i d i a a c t i v e substances by primary t i s s u e c u l t u r e s was i n v e s t i g a t e d , and the r e s u l t s are shown in Table I .

A l l of the primary

c u l t u r e s which produced C r i t h i d i a a c t i v e substances in q u a n t i t i e s than 0.6 ng/ml were considered to be a c t i v e .

greater

Of those which were a c t i v e ,

the primary c u l t u r e s o f kidney, ovary and lung c e l l s from the Syrian

252 golden hamsters were notably high.

These results suggest that organs from

this animal may be an excellent source for which to study biopterin biosynTABLE

thesis at the enzyme level. TABLE

v a r i o u s o r g a n s from the S y r i a n g o l d e n

Crithidia T h e p r o d u c t i o n of a c t i v e s u b s t a n c e s by p r i m a r y c u l t u r e s Biopterin equivalent ng/ml

Organ

_ . Condition

Liver

Complete Minus

Kidney Lung Ovary

Average of 6 c u l t u r e s Average of 3 cultures A single c u l t u r e

1.7

of 3 c u l t u r e s of 4 c u l t u r e s

Kidney

1 9 0.4

culture of S c u l t u r e s of 6 cultures

0.4 2.6 0.6

of 3 cultures of 5 cultures of 2 cultures

16 0 11 4 9 2

Biopterin

Minus

16 8 1 9 NAD(P)

Brain

0 3 NAO(P)

01

Minus

01

GTP

Plus NAD(P)H and

Minus

NAD(P)

01 NAO(P)

Complete Minus

0.9 01

GTP

Plus NAD(P)H and

1.7 01

GTP

Plus NAD(P)H and Uterus and Ovary

0 9

Complete

Complete

Lung

28 2 0 7

GTP

Plus NAD(P)H and

equivalent ng

Complete

1 3 0.5

Rat Average Lung Average Ovary Chinese hamster Kidney A single Average Lung Average Ovary Syrian golden hamster Average Kidney Average Lung Ovary Average

active

hamster

GTP

Plus NAD(P)H and

Mouse

Crithidia

s u b s t a n c e ( s ) by a d i a l y z e d s u p e r n a t a n t t r a c t i o n ot

I

Primary c u l t u r e

II

Eftect of G T P o n the s y n t h e s i s of

01 NAD(P)

01

The enzymatic synthesis of biopterin - The enzymatic synthesis of biopterin by homogenate preparations of various organs from the Syrian golden hamster was investigated.

This investigation consisted of two phases, one being

the study of the u t i l i z a t i o n of a guanine derivative and the second the u t i l i z a t i o n of D-erythrodihydroneopterin triphosphate in the enzymatic synthesis of biopterin. In an i n i t i a l experiment, dialyzed supernatant f l u i d s from homogenate preparations of l i v e r , kidney, brain, uterus and lung were individually tested for their a b i l i t y to convert GTP to biopterin-1ike substances.

The r e s u l t s ,

which are presented in Table I I , indicate that of the various organs examined, the l i v e r preparation was most active.

This a c t i v i t y is dependent

upon the presence of GTP and i s stimulated by a mixture of the pyridine nucleotides. In Table I I I , the effect of various purine derivatives, in the absence of a mixture of pyridine nucleotides, on the synthesis of biopterin-1ike sub-

253 s t a n c e s by d i a l y z e d s u p e r n a t a n t f l u i d s from a l i v e r ed.

homogenate was e x a m i n -

The r e s u l t s show t h a t w h i l e GTP and GDP were v e r y a c t i v e , the o t h e r

compounds t e s t e d showed l i t t l e

activity.

I n o r d e r to t e s t f o r the i n c o r p o r a t i o n o f r a d i o a c t i v i t y o f

or

[U-^C]GTP

[ 8 - 1 4 c ] G T P i n t o n e o p t e r i n and b i o p t e r i n , a Sephadex G-150 p r e p a r a t i o n was i n c u b a t e d w i t h a m i x t u r e o f NAD(P)H and MAD(P), a s w e l l a s 2 . 9 umoles and 2.5 yCi

[U-14C]GTP

or [ 8 - 1 4 c ] G T P a t 37° f o r

12

hours.

The r e a c t i o n was

t e r m i n a t e d and the m i x t u r e t r e a t e d by the i o d i n e p r o c e d u r e .

The

t r e a t e d m i x t u r e was a p p l i e d a t pH 1 . 0 to a Dowex-50 ( H + ) column. column was washed w i t h water and the water f r a c t i o n s e t a s i d e t i o n was d e s i g n a t e d a s Dowex-50 f r a c t i o n

I).

The

(this

frac-

The column was then e l u t e d

w i t h 1 N NH4OH ( t h i s f r a c t i o n was d e s i g n a t e d Dowex-50 f r a c t i o n pH o f the Dowex-50 f r a c t i o n I

iodine-

II).

The

( t h e water wash) was a d j u s t e d t o 8 . 0 w i t h

2 M T r i s base, a l k a l i n e phosphatase

(1 mg per ml o f f r a c t i o n

I ) was a d d e d ,

and t h e m i x t u r e was i n c u b a t e d f o r 1 hour a t 37°C in the d a r k .

The m i x t u r e

was a g a i n a c i d i f i e d t o pH 1 , a p p l i e d to a n o t h e r Dowex-50 column ( H + ) , and the column washed w i t h water and the water f r a c t i o n d i s c a r d e d . 50 column was then e l u t e d w i t h 1 N NH4OH.

This

The p t e r i n s e l u t e d from each

Dowex-50 column were p u r i f i e d i n d i v i d u a l l y by the use o f

ECTEOLA-Sephadex

(OH") and ECTEOLA-Sephadex pH 7 . 0 column chromatography a s d e s c r i b e d viously.

Dowex-

From the ECTEOLA-Sephadex pH 7 . 0 c o l u m n , a s i n g l e blue

pre-

fluore-

s c e n t band was e l u t e d w i t h water and a s s a y e d f o r C r i t h i d i a a c t i v i t y .

The

e l u a t e was c o n c e n t r a t e d and a p p l i e d w i t h c a r r i e r b i o p t e r i n and D - e r y t h r o n e o p t e r i n t o c e l l u l o s e TLC s h e e t s .

The s h e e t s were d e v e l o p e d i n

Solvent

B and the s p o t s c o r r e s p o n d i n g to b i o p t e r i n and D - e r y t h r o n e o p t e r i n were removed, e l u t e d w i t h w a t e r , c o n c e n t r a t e d and each a p p l i e d t o a n o t h e r TLC sheet.

A two-dimensional

chromatography was performed by d e v e l o p i n g

each

254sheet, first in Solvent E and then Solvent A.

Finally, the spots corres-

ponding to authentic D-erythroneopterin and biopterin were removed and assayed for radioactivity. TABLE III Effect of certain purine compounds on the enzymatic synthesis of Crithidia active substance(s) by Syrian golden hamster liver homogenate preparation Compound

Biopterin Equivalent ng

GTP

37.5

GDP

25

GMP

2.5

Guanosine

2.5

ATP

3.6

None

2.5

TABLE IV Products froin « N«opt*rin

Bioptarin

0.1 23

1,40« IN

80

0.1 24

0 T

6

< tram tha ECTKHA-Sephedu pH 7.0 Chromatography. * Spots corrtspondinQ to avtJwiitlc waoptariw M d Moptarln from tho Anal TLC.

The results of these experiments are presented in Table IV. in the reaction mixture were resolved into two fractions.

The products Dowex-50 frac-

tion I of the [U-^C]GTP reaction mixture contained very little Crithidia active substances but contained a high level of radioactive neopterin. However, from the Dowex-50 fraction II, purified fractions contained a high level of Crithidia active substances but a low level of radioactive neopterin and biopterin.

From the [8-l^c]GTP experiments, the Dowex-50

I fraction contained very little Crithidia activity and no radioactive neopterin.

On the other hand, the Dowex-50 II fraction contained Crithidia

active substances but very little radioactive neopterin and biopterin. These results suggest that a phosphorylated product is present in the Dowex-50 fraction I, which after a phosphatase treatment was recovered as neopterin.

The results also suggest that since neopterin is found in the

Dowex-50 II fraction, the enzyme preparation contains some phosphatase activity.

Furthermore tnese data indicate that the enzyme preparation also

synthesizes biopterin.

The evaluation of these results supports the

255 hypothesis for a ring-opening reaction of GTP. For purposes of obtaining additional data as to the identity of the products from GTP, a fraction obtained from an Ultragel AcA-34 chromatographic purification of a liver homogenate preparation was incubated with GTP for 3 hours.

The reaction mixture was oxidized- by the iodine proce-

dure and applied to a Sephadex G-25 column.

The fluorescent peak frac-

tions were pooled and a portion treated with alkaline phosphatase. Samples of the phosphate treated and untreated fractions were chromatographed on TLC sheets in five solvent systems.

The r e s u l t s , which are

presented in Table V, show that the mobility of the fluorescent material produced by a l i v e r preparation before the phosphatase treatment is similar to the iodine-treated product derived from GTP prepared from a L_. plantarum preparation.

On the other hand, the alkaline phosphatase

treated fraction contained a fluorescent material which migrated as neopterin. for

This material was then assayed for i t s growth promoting a c t i v i t y fasciculata.

The a c t i v i t y of the product which i s shown in Fig. 3

i s similar to authentic D-erythroneopterin.

BioPTERiN.ng/iube 010

005

TABLE

015

0 20

0 25

V

Thin-layer chromatography ol the reaction product

Compound

A

Solvents B

Rt Values G D

06 E

Biopterin

0 52

0.22

0.41

0.39

0.44

Erythro neopterm

0.50

0 09

0 28

0.25

0.28

Threo neopterin

0.46

0.08

0.20

0.21

0.24

Product from GTP with L. p. preparation"

0.85

0.0

0.12

0.03

0.18

Before alkaline phosphatase

0.85

0.0

0.12

0.03

0.17

After alkaline phosphatase

0.50

0.0«

0.28

0.28

0.28

Product from GTP with liver preparation

| o Of) ID 1< (J 0 4 Z OD oc i/i m

as

"Solvent systems sre described in materials end Methods. "L plantarum preparation

0

5

10 15 120 25 30 D- ERYTHRO-MFOPTFBIN. ng/tube

Fig. 3. C. fasciculata growth promoting a c t i v i t y of a dephosphorylated product from bI P.

35

256 For initial studies concerning the investigation into the enzymatic synthesis of biopterin from radioactive D-erythrodihydroneopterin triphosphate by organ homogenates of Syrian golden hamsters, crude homogenates composed of 25 percent (wet weight by volume) of brain, liver, lung or kidney were prepared. was performed.

For the assay for synthesis, a two-stage experiment

In the first stage

(1 yCi and 0.6 ymole) was

incubated in Tris-KCl buffer, with an enzyme preparation from L_. plantarum, which synthesized D - e r y t h r o d i h y d r o n e o p t e r i n triphosphate.

At 2 hours

(second stage), potassium phosphate buffer, pH 6.8, MgSCty, a mixture of NAD, NADH, NADP and NADPH, and crude homogenate were added to the stage 1 mixture.

The reaction mixture was incubated for 5 hours at 37° in the

dark and the reaction terminated by adding 0.2 ml of 1 N HC1.

After oxida-

tion with iodine, pterins were analyzed by a column chromatography and TLC procedure as previous described. The results which are presented in Table VI indicate

that radioactive

biopterin was produced by the combined homogenate, and that this production was dependent upon the addition of L_. plantarum enzyme preparation to stage one.

Of the various organ homogenates, the kidney prepara-

tion was most active.

The loss of activity seen upon using boiled liver

preparations in stage two indicates that heating inactivates liver preparations and abolishes production of radioactive biopterin from bacterial enzyme-treated GTP. In another experiment, the effect of GTP on dialyzed supernatant fractions of the various organ preparations in the two-stage incubation system was determined by the Crithidia assay. was omitted.

For this assay the iodine treatment

These results, presented in Table VII, indicate that the

TABLE VII TABLE

VI

Production of Cnthidiê active swbstance(s) from encyme-treated GTP by a dialyied supernatant fraction of various organs from Syrian golden hamster

Incorporation ol radioactivity o( enzyme-treated (U- 1 4 C)GTP into btopterin by crudo homogenste* of various organ» from Syrian golden hamster Condition Experiment 1 2

Stage 1

Condition S U g e 2**

Total CrtthWë Activity in Btopterin Equivalent, ng per assay

dpm

Stage 1 '

Stage 2 "

0 hr

Complaît

Kidney

09 0.3

Complete

Combined homogenate***

3,340

w/o L p enxyme

2B

w/o GTP

Kidney

Complete

Combined homogenate*** Liver

2,419

Complete

Liver

07

Complete

Boiled liver

Specific Activity ng/mg protein

9 hrs

9 hrs

990

12« -

09

0

w/o GTP

Liver

09

09

43 -

11,BBS

Compiete

Brain

w/o GTP Complete

Brain

07 09

299 09

99 -

Lung

09

299

34

w/o GTP

Lung

09

Complete

Kidney

Complete

Brain

1,244

Complete

Lung

1,122

Stage 1 contains GTP and L plantarum triphosphate synthetase fraction

'

D*eryfhrodihydroneoptertn

Stage 2 contains organs which were homogenized to give 2 5 % wet weight per volume and 0 4 0 ml of each preparation was used per assay. The boiled liver was prepared by heating the homogenate preparation at 100° for 3 0 seconds

299

0.7

-

* Stage 1 contains L pfenferam O-eryfArodHiydroneopterln triphosphate synthetase fraction " Dialyzed supernatant fractions were used in stage 2

Combined homogenate was a mixture of brain, liver, lung and kidney.

dialyzed supernatant fractions of kidney, l i v e r , brain and lung were all active in the production of Crithidia active substance(s) and that the a c t i v i t y was dependent upon the addition of GTP to stage one. organs tested, the kidney was the most active.

Of a l l the

The specific a c t i v i t y of

brain was also high, because of the low protein content of this organ. Since the dialyzed supernatant fraction of kidney was most active, subse-

O : FLUORESCENT • • BIOPTERIN

20 10

20 FRACTION

SUBSTANCE

40

BIOPTERIN

i f f M i

NUMBER

Fig. 4. Elution profile of fluorescent substance 1 from pH 7 ECTEOLASephadex column. Fluorescence was measured after a 10-fold d i l u t i o n . Fig. 5. Dose response of C^. fasciculata to fluorescent substance 1 and biopterin.

I

258 quent experiments were carried out using t h i s kidney preparation. In order to obtain more information on the reaction product(s), a largescale experiment was performed by increasing the components of the reaction mixture of stage one and stage two, f i v e - f o l d .

The terminated reac-

tion mixture, which was treated with iodine, was processed by the three column chromatographic procedure as previously described.

A single blue

fluorescent band which was eluted with water, at about 4 to 5 times the bed volume, from the final chromatographic step was concentrated and streaked on a thin-layer chromatogram.

The chromatogram was developed in Sol-

vent B, without the addition of carrier biopterin.

The fluorescent fraction

was resolved into three bands (fluorescent substances 1, 2 and 3), each of which was found to be radioactive.

The band corresponding to biopterin

(fluorescent substance 1) was scraped, eluted with water, and the eluate was rechromatographed in Solvent A.

The chromatogram showed only a single

fluorescent band and coincident single radioactive area which correspondThe fluorescent band was subjected to the Dowex-50 (H + )

ed to biopterin. TABLE

VIII TABLE

T h i n - l a y e r c h r o m a t o g r a p h y of the r e a c t i o n p r o d u c t s

X

Tha affact ol a mixtiira of oxidized and raducad pyrldlna nudaotldas and tha compaflaon of tha apacrfic radioactivity

S o l v e n t s *, Rf V a l u e s Compound

A

Biopterin

67

34

Erythroneopterin

64

7/rreoneopterin Pterin

B

C

F

G

D

E

59

.55

59

10

55

32

44

48

25

60

08

50

30

42

40

15

51

.33

52

52

57

6-Hydroxymethylpterin

51

29

52

51

52

Fluorescent substance 1

67*

34*

59*

55*

59*

Fluorescent substance 2

64*

10*

55*

32*

44*

Fluorescent substance 3

s r

27*

53*

53*

53*

Condition Expartmant

.48*

1

2

Staga 1

Staga 2

Complata

Complata

Complata

w/o pyndina nudaotldas

Complata

Complata

(U-14C)0TP

(U- t 4 C)D-Err«»rodlhydronaoptarln trlphoaphate

3 0 fimolas

2 9 jjmolas

3,310 dpm/nmola

3,050 dpm/nmola

Bioptarin 11,659 dpm 2 2 5 dpm

1 1 8 nmolas 2.BB0 dpm/nmola

.25'

•Radioactive " S o l v e n t s y s t e m s a r e d e s c r i b e d in M a t e r i a l s a n d M e t h o d s .

and the two ECTEOLA-Sephadex column chromatograhic procedures and assayed for radioactivity, fluorescence and Crithidia a c t i v i t y .

These results

which are presented in Figures 4 and 5 indicate that the specific radioa c t i v i t y of the fractions collected from the final pH 7.0 ECTEOLA-Sepha-

259 dex column was constant and that the growth-promoting a c t i v i t y for Crithidia was similar to biopterin.

The fluorescent fractions were pooled

and subjected to thin-layer chromatography.

The results (Table V I I I )

demonstrate that a coincident fluorescent and radioactive spot migrated identically to authentic biopterin in each of the five solvent systems. Fluorescent substance 2 was also purified by the three column chromatographic steps and subjected to thin-layer chromatography.

A single fluore-

scent and radioactive spot migrated identically to authentic erythroneopterin in each of the seven solvent systems. Although fluorescent substance 3 migrated similarly to 6-hydroxymethylpterin, at present we are not absolutely certain of i t s identity. In Table IX, the results of several experiments are summarized.

In experi-

ment 1, the addition of a mixture of oxidized and reduced pyridine nucleotides i s shown to be required by the dialyzed kidney preparation.

In ex-

periment 2, i t can be seen that the specific radioactivity of the isolated biopterin compares favorably with that of the isolated D-erythrodihydroneopterin triphosphate which was produced by GTP in stage 1. Reversal of 4-benz.ylthio-7-azabiopterin (azabiopterin) inhibition with biopterin in Chinese hamster ovary cell cultures - Cultures of Chinese hamster ovary c e l l s have been shown to produce biopterin, however, i t has yet to be determined whether this biopterin, or one of i t s reduced forms, i s required by these cell cultures for growth.

As an approach to the study

of this problem we have initiated experiments designed to examine the u t i l i zation of biopterin in Chinese hamster ovary c e l l s in culture.

In pre-

liminary experiments we examined the effect of the biopterin analogue, 4benzylthio-7-azabiopterin on the efficiency of plating and generation time

260 of the Chinese hamster ovary c e l l s growing in Ham's F-12 medium containing 10% charcoal-treated fetal c a l f serum.

This biopterin analogue has been

found by Dr. John McCormack to have i n h i b i t o r y a c t i v i t y against r a t l i v e r phenylalanine hydroxylase.

We found a s i g n i f i c a n t reduction in p l a t i n g

e f f i c i e n c y and prolongation of generation time of c e l l s when azabiopterin was included in the cell culture medium.

In subsequent experiments, the

c e l l s were grown in F12-D medium (17) containing 10% c h a r c o a l - t r e a t e d , d i a lyzed fetal c a l f serum.

As a measure of c e l l growth,

(4,5-^H)-L-leucine

was added to the medium and the amount of r a d i o a c t i v i t y incorporated into c e l l u l a r protein was determined.

Cell cultures containing only F12-D medo

ium, charcoal-treated, dialyzed fetal c a l f serum and controls.

H-leucine served as

To certain of the normally growing c e l l c u l t u r e s , azabiopterin

was added at a concentration of 6 x 10 - 5 M.

To other c e l l cultures was add-

ed, in addition to a z a b i o p t e r i n , either biopterin (100 ng/ml) or dihydrobiopterin (100 ng/ml).

Cell c u l t u r e s containing only biopterin or dihydro-

b i o p t e r i n , in these same concentrations, a l s o served as c o n t r o l s . s u l t s from one experiment are shown in Table X.

The r e -

From t h i s preliminary i n -

v e s t i g a t i o n i t appears that azabiopterin p a r t i a l l y i n h i b i t s c e l l

prolifera-

tion and that b i o p t e r i n , but not d i h y d r o b i o p t e r i n , can reverse t h i s TABLE X R e v e r s a l of 4 - B e n z y l t h i o - 7 - a z a b i o p t e n n (Azabiopterin) Inhibition with Biopterin in C H O Cell Cultures Addition

Incorporation of (4,5 3 H ) L - L e u c i n e dpm

%

None

50,255

100 0

Dihydrobiopterin

56,658

112 7

Biopterin

41,579

82 7

Azabiopterin

26,338

52 4

29,021

57 7

47,370

94 2

Azabiopterin + Dihydrobiopterin Azabiopterin + Biopterin

inhibi-

261 tion.

Although the s i t e of action of azabiopteriri i s , as yet, unknown, i t

is tempting to speculate that i t may be inhibiting a biopterin requiring enzyme(s) whose function i s necessary for normal cell growth. Discussion The results which were recently obtained with Chinese hamster ovary c u l tures, primary tissue cultures and the enzyme preparations of organs from the Syrian golden hamster indicate that biopterin i s synthesized from GTP. The reactions involving GTP may be identical in mammalian systems as those that occur in bacterial systems.

These reactions include an imidazole

ring-opening step, the loss of carbon atom 8 from the purine r i n g , and ring closure resulting in the formation of D-erythrodihydroneopterin t r i phosphate.

The reactions involving the side-chain of D-erythrodihydroneo-

pterin triphosphate in i t s conversion to biopterin, are not as yet known; an exception being the possible involvement of a reaction which converts sepiapterin to dihydrobiopterin.

Presently we are considering the p o s s i -

ble alterations of the side-chain to include dephosphorylation, reduction, oxidation and dehydration steps. Acknowledgments This work was supported by grant BC-107C from the American Cancer Society and grant AM16622 from the National Institute of A r t h r i t i s , Metabolism and Digestive Diseases and grant DE-02670 from the National Institute of Dental Research, National Institutes of Health.

The biopterin analogue,

4-benzylthio-7-azabiopterin was synthesized by Dr. E. C. Taylor and his colleagues at Princeton University, Princeton, New Jersey and kindly provided by Dr. John J. McCormack, J r . , University of Vermont, Burlington, Vermont.

262 References

1.

Brown, G. M.:

The biosynthesis of pteridines.

Advan. Enzymol. 35,

35-76 (1971 ). 2.

Shiota, T.:

The biosynthesis of folic acid and 6-substituted pteri-

dine derivatives. 3.

Comp. Biochem. 21_, 111-152 (1971 ).

Plowman, J., Cone, J. E., and Guroff, G.:

Identification of D-er.ythro-

dihydroneopterin triphosphate, the first product of pteridine biosynthesis

Commamonas sp. (ATCC 11299a).

J. Biol. Chem. 249., 5559-

5564 (1974). 4.

Kobashi, M., and Iwai, K.:

Enzymatic synthesis of the Crithidia

factors in cell-free extracts of Serratia indica.

Agri. Biol. Chem.

36, 1695-1700 (1972). 5.

Kidder, G., and Dewey, V. C.:

A new pteridine from Tetrah.ymena.

J.

Biol. Chem. 243, 826-833 (1968). 6.

7.

Levy, C. C.:

Pteridine metabolism in the skin of the tadpole, Rana

catesbeiana.

J. Biol. Chem. 239, 560-566 (1964).

Sugiura, K., and Goto, M.:

Biosynthesis of pteridines in the skin of

the tadpole, Rana catesbeiana. 8.

Fukushima, T.:

Biosynthesis of pteridines in the tadpole of the bull-

frog, Rana catesbeiana. 9.

J. Biochem. 6£, 657-666 (1968).

Arch. Biochem. Biophys. 139., 361-369 (1970).

Brenner-Hoizach, 0., and Leuthardt, F.:

Die biosynthese der pterine,

IV Die berkunft der scitenkette in biopterin.

Z. Physiol. Chem. 348,

605-606 (1967). 10.

Goodfriend, T. L., and Kaufman, S.: folic acid antagonists.

11.

J. Clin. Invest. 40, 1743-1750 (1961).

Fleming, A. F., and Broquist, H. P.: ciency.

Phenylalanine metabolism and

Biopterin and folic acid defi-

Amer. J. Clin. Nutr. 20, 613-621

(1967).

263 12.

Pabst, W., and Rembold, H.: saugetierorganismsus, II.

13.

Uber das verhalten des biopterins im Z. Physiol. Chem. 344, 107-112 (1966).

Fukushima, T., and Shiota, T.:

Biosynthesis of biopterin by Chinese

hamster ovary (CHO K1) cell culture.

J. Biol. Chem. 249, 4445-4451

(1974). 14.

Buff, K., and Dairman, W.: of mouse neuroblastoma.

15.

Ham, R. G.:

Mol. Pharmacol. 11_, 87-93 (1975).

Clonal growth of mammalian cells in a chemically de-

fined, synthetic medium. 16.

Biosynthesis of biopterin by two clones

Proc. Nat. Acad. Sci. 53, 288-293 (1965).

Shiota, T., Pal umbo, M. P., and Tsai, L.:

A chemically prepared

formamidopyrimidine derivative of guanosine triphosphate as a possible intermediate in pteridine biosynthesis.

J. Biol. Chem. 242,

1961-1969 (1967). 17.

Kao, F. and Puck, T. T.:

Genetics of somatic mammalian cells, VII.

Induction and isolation of nutritional mutants in Chinese hamster cells.

Proc. Nat. Acad. Sci. 60, 1275-1281

(1968).

264 DISCUSSION Cremer-Bartels: Did you cultivate the cells over the period of a whole year ? Were there seasonal variations ? Shiota:

These primary cultures were kept for 2 - 3 weeks,

G.M.Brown: Is dihydroneopterin triphosphate converted enzymatically to a product m the absence of pyridine nucleotides ? Shiota: We don't know, however, I might add that from preliminary evidence, the phosporylated form is not converted to biopterin. Only by future work, the purification and separation of the enzymes, can we be able to answer your question. Nair: To avoid confusion on the nomenclature of these compounds, it would have been easi&r if you specify the absolute configuration of these molecules in the (R) and (S) convention. Would you consider this proposal ? Shiota: Yes. We would consider it. Buff: The first step of the reaction (GTP -*• D-erythro-dihydroneopterintriphosphate) seems to take place most efficiently in homogenates derived from liver cells. The second step seems to occur most efficiently in kidney preparation (PH-Neo-triphosphate-»- biopterin). Is there any physiological significance for this phenomenon ? Shiota: At the moment, we have no explanation for the different activities found in liver and kidney homogenate preparations from the Syrian golden hamsters. Rembold: Is there biopterin or neopterin synthesis by mitochondria or subfractions ? Shiota: We did not test any sub-cellular fractions, Rembold: You worked with cell cultures. Do organs synthesize biopterin in the same rate as cells ? Shiota: We don't know the answer to this question.

The Synthesis of Pterins Catalyzed by Enzymes from Drosophila melanogaster G.M. Brown and C.L Fan

INTRODUCTION Although the enzymatic pathway for the synthesis of pterins in bacteria has been elucidated and each enzyme has been investigated relatively intensively (1), whether or not similar enzymatic reactions occur in animals has remained unclear.

In deciding which animal system to study the enzy-

matic synthesis of pterins, we chose the fruit fly, Drosophila melanogaster, for our studies because this animal is known to make relatively large quantities of a variety of pterins (2), including the drosopterins, sepiapterin, pterin, xanthopterin, isoxanthopterin and biopterin.

Thus, it

would appear that the fruit fly should be a rich source of the enzymes needed to produce these substances. Previous evidence (3,4) has indicated that in insects, like bacteria, a guanine compound is probably a precursor of pterins.

Since the first en-

zyme concerned with the synthesis of pterins in bacteria is known to be GTP cyclohydrolase, we decided to look for this enzyme in Drosophila.

We

were successful in this endeavor and the results presented below describe some of the properties of the enzyme and the reaction it catalyzes. DEVELOPMENT OF GTP CYCLOHYDROLASE IN Drosophila melanogaster Homogenates were made of eggs, larvae, pupae, and adult flies during the various stages of development and GTP cyclohydrolase activity of each 14 homogenate was measured with the use of the assay for [ C]formate produc14 tion from [8-

C]GTP developed earlier (5).

The results given in Fig. 1

show that activity appears at two stages of development: a relatively small peak at the time of pupariation and a much larger amount as the pupae approach the time of eclosion (emergence of the adult fly). eclosion, enzyme activity drops rapidly as the adult ages.

After

266

Doys After Oviposition

Fig. 1. GTP cyclohydrolase activity in Drosophila melanogaster during development through the larval and pupal stages into adult flies. H = hatching; P = pupariation; E = eclosion from pupae to adults. The possible correlation of the development of GTP cyclohydrolase activity and the appearance of various pterins during development was sought.

The

results presented in Fig. 2A show that at the time of pupariation the appearance of isoxanthopterin, pterin, and sepiapterin is coincident with the development of enzyme activity (i.e., the first peak of enzyme activity in Fig. 1).

Fig. 2B shows that development of the main peak of activi-

ty evident in Fig. 1 follows along with the appearance of the drosopterins, the major pterins found in Drosophila.

These observations strongly sug-

gest that GTP cyclohydrolase is directly involved in the biosynthesis of pterins in the fruit fly. One-day old (male and female) flies (50 of each sex) were dissected to separate the heads from the thoraxes and abdomens.

These tissues were

homogenized and the resulting homogenates were analyzed for GTP cyclo1

I

Enzyme

I I

Enzyme Activity

Activity

Drosopterin

Isoxanthopterin Pterin

ti

Sepiapterin

30 _

Isoxanthopterin

3 ®

Pterin Sepiapterin

2 . 0 «>

/ x

Hi

ùr'

_L I

2

_L 3

Days A f t e r

4

_L 5 6

Oviposition

I 0

2

3

4

Days After Puporiation

Fig. 2. Correlation of development of GTP cyclohydrolase activity with production of pteridines. The production of pteridines was measured in arbitrary units of fluorescence.

267

Localization of CTP Cyclohydrolase Activity m

Young Adults

GTP cyclohydrolase Tissue

Table I.

Sex

activity (per animal)—

Intact fly

Female

0.161

Intact fly

Male

0.143

Head

Female

0.150

Head

Male

0.137

Thorax plus abdomen

Female

0.040

Thorax plus abdomen

Male

0.027

— Enzyme activity is expressed as nmoles of formate released from GTP per hour.

hydrolase activity.

The data in Table I indicate that most (about 80%) of

the enzyme activity is present in the head.

This finding is not surprising

since most of the pterins are present in Drosophila as eye pigments and it therefore seems reasonable to expect that synthesis occurs by enzymes found in the head.

The data of Table I also indicate that the amount of

GTP cyclohydrolase activity found in females is somewhat higher than that found in males, probably because females are somewhat larger than males. We have tested 18 separate mutants with variations in eye color owing to defective synthesis of pterin eye pigments.

These include several whose

phenotype is white eyes (i.e., no drosopterin or sepiapterin) as well as some with abnormal eye colors (e.g., one that contains sepiapterin, but no drosopterin).

We have found that all of these mutant flies contain GTP

cyclohydrolase with only minor variations in specific activities.

Thus,

none of these mutations appear to effect the synthesis of a fully active GTP cyclohydrolase.

PURIFICATION AND PROPERTIES OF GTP CYCLOHYDROLASE FROM Drosophila Since late pupae and young adults contain the largest amounts of enzyme Purification of GTP Cyclohydrolase From Extracts of Drosophila melanogaster

Enzyme preparation

Total activityunits

Table II.

Specific activity— units/mg protein

Crude extract

5600

3.1

Ammonium sulfate fraction

3010

10.8

Fractions from Sepharose 48 column

1260

50 0

— One unit is the amount of enzyme needed for production of one nroole of formate per hour from GTP [200 yM) at pH 7.5 and at 42°.

268

(see Fig. 1), we devised a procedure for purification of the enzyme from this source.

For this purpose, a mixture (approximately 60 gm) of late

pupae and young adults (no more than one day old) was homogenized and the resulting material was subjected to centrifugation to remove insoluble material.

The soluble material (the "crude extract") was subjected to the

fractionation scheme summarized in Table II.

The enzyme used in the re-

mainder of the work to be reported below was the preparation obtained by this procedure. An investigation of the substrate specificity of the enzyme has revealed that GDP and dGTP are used approximately 1.8% and 1.5%, respectively, as well as GTP.

Guanosine, GMP and ATP were not used at all.

ATP and dGTP,

when provided in concentrations five-fold greater than GTP, inhibited by 24% and 72%, respectively, the production of formate from carbon 8 of GTP. The K^ value for GTP has been estimated at 22 yM from a double reciprocal (Lineweaver-Burk) plot. The enzyme functions optimally at pH 7.8 and at 42°.

The activity of the

enzyme is not affected by the addition of salt (NaCl or KC1) at concentrations as high as 10 mM.

However, its activity is severely inhibited by

the following divalent cations: Mg

2+

, Mn

2+

, Zn

2+

, or Ca

2+

.

The molecular

weight of the enzyme has been estimated at 345,000 by its behavior on a column of Sepharose 4B calibrated with the following standard proteins of known molecular weight: bovine thyroglobulin (669,000), rabbit muscle aldolase (158,000), ovalbumin (45,000), and ribonuclease A (13,700). PRODUCTS Formate was identified as one product of the action of GTP cyclohydrolase from Drosophila by methods described in an earlier paper (5).

This in14 volved^the conversion of radioactive formate (produced from [8- C]GTP) to formyl hydroxamate and its identification as this material by comparison of its paper chromatographic characteristics with that of standard formyl hydroxamate. By analogy with the bacterial systems, we expected that the other product of the reaction would be dihydroneopterin triphosphate

(H.-neopterin-PPP).

269

nm •Fig. 3. Absorption spectrum of the product of action of GTP cyclohydrolase from Drosophila melanogaster. To obtain evidence bearing on this point, we incubated the enzyme with [ U - ^ C J G T P and subjected the incubated reaction mixture to chromatography on DEAE-cellulose substrate.

(6) to separate the putative product from the unreacted

The fractions containing the radioactive product were combined

and an absorption spectrum was taken of the material.

The resulting spec-

trum, given in Fig. 3, is virtually identical with that of standard neopterin, with maxima at 330 nm and 275 nm.

Confirmation of its identity

as H^-neopterin was obtained with the observation that the dephosphorylated product (formed by treatment with alkaline phosphatase) behaved on paper chromatographic analysis as standard D-erythro-H -neopterin.

Distance from O r i g i n , cm

D i s t a n c e from O r i g i n , cm

Fig. 4. Paper chromatography of incubated reaction mixtures prepared with either [8- 1 4 C,y- 3 ^P]GTP (A) or [ U - 1 4 C , y - 3 2 P ] G T P (B). The solvent system was prepared by mixing 1 liter of 0.1 M sodium phosphate (pH 6.8), 600 gm of ammonium sulfate, and 20 ml of 1-propanol. Development was by the descending technique. The chromatograms were inspected under ultraviolet light for fluorescent zones and then each chromatogram was cut horizontally into 1-cm strips and each strip was analyzed for 1 4 C and radioactivity in a liquid scintillation counter in the presence of Aquasol (5 ml) and water (1.7 ml).

270 The evidence cited above indicates that the dephosphorylated product is D-erythro-H^-neopterin, but it does not provide information about whether or not the product contains phosphate residues.

To provide evidence 14 32 about this question, the enzyme was incubated with either [U- C,y- P]GTP or [8- 14 C,y- 32 P]GTP and the incubated mixtures were subjected to paper

chromatography as described in the legend of Figs. 4A and 4B. Figs. 4A 32 and 4B show that a P-containing compound (migration of 12-18 cm) was 32 produced from [y-

P]GTP.

This product did not contain carbon 8 of the

original GTP provided as substrate; instead, carbon 8 appeared on the chromatogram as a radioactive peak (Fig. 4A) (migration of 31-36 cm) in the general area formate expected to beenzymatic found. product The results sented in Fig. 4B where show that the is 32 P-containing also precon14 14 C radioactivity provided in the reaction mixtures as [U- C]GTP. 14 Again, a C-containing compound migrated in the general area of migration 32 of standard formate. The P peak at around 40 cm on both chromatograms tains

results from an unidentified impurity present in the labeled GTP. It 32 should also be mentioned that the zone of migration of the P-containing enzymatic product was also blue fluorescent, as would be expected if the product were dihydroneopterin or phosphoesters of this compound.

Since

the y-phosphate of the GTP substrate was retained in the enzymatic product, we conclude that the product was a triphosphoester.

This, along

with the spectrophotometry and paper chromatographic evidence, leads to the conclusion that the enzyme catalyzes the production of D-erythro-H^neopterin-PPP, and in this respect is similar to GTP cyclohydrolase I from Escherichia coli. DISCUSSION GTP cyclohydrolase from Drosophila is very similar to the bacterial enzyme in: (a) its relatively high molecular weight; (b) its substrate specificity; (c) its pH and temperature optima; and (d) the products of its action.

Since activity develops in Drosophila at about the same time that

pterins appear, we conclude that GTP cyclohydrolase is involved in the biosynthesis of these pterins.

Since neither H^-neopterin-PPP, the pro-

duct of action of the enzyme, nor the dephosphorylated compound (l^-neopterin or neopterin) is one of the pterins that has been isolated from

271 Drosophila, it would appear that this enzymatic product is immediately metabolized further in Drosophila to give ultimately the pterin end products (the drosopterins, sepiapterin, etc.)-

Thus, H^-neopterin-PPP would

appear to be a key intermediate in the biosynthesis of other pterins just as it is in bacteria.

The enzymes that metabolize H^-neopterin-PPP to

yield the pterin end products in Drosophila have not yet been studied, but it would now appear to be feasible to study these systems at the enzymatic level and thus to obtain information toward an understanding of the biosynthesis of these interesting pterin compounds. Acknowledgements. The authors thank Professor Linda Hall of the Biology Department at the Massachusetts Institute of Technology for her help and advice in the growing and handling of the animals used in these studies. These investigations were supported by research grants from the National Institutes of Health of the U.S. Public Health Service (grant number AM03442) and the National Science Foundation (grant number GB33929X1).

REFERENCES 1. G.M. Brown, J. Yim, Y. Suzuki, M.C. Heine and F. Foor: The enzymic synthesis of pterins in Escherichia coli. This Symposium, pp. 2. Ziegler, I. and Harmsen, R.: The biology of pteridlnes in insects. Adv. Insect. Physiol. 6, 139-203 (1969). 3. Watt, W.B.: Pteridine biosynthesis in the butterfly Colias eurytheme. J. Biol. Chem. 242, 565-572 (1967). 4. Goto, M. and Sugiura, K.: Biosynthesis of pteridines in Drosophila melanogaster and Rana catesbeiana. Methods in Enzymology 18B, 746761 (1971). 5. Burg, A.W. and Brown, G.M.: The biosynthesis of folic acid VIII. Purification and properties of the enzyme that catalyzes the production of formate from carbon atom eight of guanosine triphosphate. J. Biol. Chem. 243, 2349-2358 (1968). 6. Suzuki, Y. and Brown, G.M.: The biosynthesis of folic acid XII. Purification and properties of dihydroneopterin triphosphate pyrophosphohydrolase. J. Biol. Chem. 249, 2405-2410 (1974).

272 DISCUSSION Schwinck: Flies after eclosion show still a very strong synthesis of drosopterins and other pteridines, including sepiapterin, for many days up to 10 days or more - why does your enzyme activity decrease so fast after eclosion if it makes the precursor ? G.M.Brown: 1 have no obvious answer axcept to say that the patterns we reported for enzyme activity are reproducible. McCormack: Do any of the pterin derivatives (e.g. isoxanthopterin, etc.) inhibit the cyclohydrolase system of Drosophila ? Could such inhibitory effects complicate determinations of enzyme activity at times when large amounts of pterins are present ? G.M.Brown: Since the crude extracts we use are heavily contaminated with such pterins, I would say that no such inhibitions are evident. Descimon: Have you obtained other pteridines than sepiapterin from incubation of I-^-neopterin-triphospbate with Drosophila head extracts ? G.M.Brown:

No.

Descimon: Have you observed a 2',3-cyclic phosphate of Q-erythro or L-threo neopterin ? G.M.Brown: No. I believe such cyclic phosphates are artefacts. Ferone: Do fruit flies make folates de novo or require them in the diet ? Mosquitos are inhibited by sulfonamides and presumably make folate de novo. Perhaps pteridine hiosynthesis in flies goes on to folate production as well as pigment production. G.M.Brown: We haven't yet determined whether or not folate is needed by Drosophila. Hackstein: You listed an increase in isoxanthopterin content after pupation which fits very well to the increase of cyclohydrolase activity. This would not be quite convincing since it is the time at which individuums cease excreating isoxanthopterin. Did you look at the isoxanthopterin excreated by larvae ? G.M.Brown:

No.

27^ e x c r e t i o n . A l t e r n a t e l y , m a m m a l s c o u l d b e c a p a b l e of d e n o v o

biopterin

s y n t h e s i s . T h e p u r p o s e o f o u r i n v e s t i g a t i o n s w a s to d e t e r m i n e

if m a m m a l s

c o u l d i n d e e d s y n t h e s i z e b i o p t e r i n dji n o v o .

The b i o s y n t h e t i c terial systems

p a t h w a y for p t e r i n s h a s b e e n i n t e n s i v e l y s t u d i e d i n b a c -

(8). T h e i n i t i a l s t e p in the b a c t e r i a l p a t h w a y

is the

a g e of the i m i d a z o l e r i n g of g u a n o s i n e t r i p h o s p h a t e a n d the l o s s of 8 as formate,

followed by rearrangement and ring closure

dihydroneopterin

triphosphate

cleavcarbon

to f o r m D - e r y t h r o -

(9). A s s u m i n g t h a t the m a m m a l i a n

pathway,

if it e x i s t e d , m i g h t b e s i m i l a r to t h a t of b a c t e r i a , w e h a v e a t t e m p t e d demonstrate

in m a m m a l i a n s y s t e m s

the i n c o r p o r a t i o n of the

to

radioactivity

f r o m s p e c i f i c a l l y l a b e l e d g u a n o s i n e , g u a n i n e o r g l y c i n e into

biopterin.

Germ free, 8 w e e k old, female, CDF strain rats w e r e obtained from R i v e r , W i l m i n g t o n , M a s s . For a p e r i o d o f o n e w e e k p r i o r to the

Charles

experiment

these animals w e r e maintained under sterile conditions either on a diet of p u r i n a lab c h o w o r o n a s y n t h e t i c b i o p t e r i n free d i e t of the composition:

c o r n s t a r c h 45

following

(grams p e r 1 0 0 g r a m s o f d i e t ) , a m i n o a c i d s

( equal parts of valine, leucine, isoleucine, lysine, arginine, methionine, histidine,

tryptophan and phenylalanine),

threonine,

coconut oil

s u c r o s e 15, C a C 0 3 1 . 6 , s o d i u m c i t r a t e 0 . 8 , K H 2 P 0 ^ 0 . 8 , K C 1 0 . 4 8 , 0.2, M g C l 2 - 6 H 2 0 0.2, C u S 0 4 - 5 H 2 0 0.004, M n S 0 4 and

25

10, inositol

0 . 0 1 , KI 0 . 0 0 8 , F e C l 3

0.02

(in m i l l i g r a m s p e r 100 g r a m s o f d i e t ) t h i a m i n e - H C l 0 . 5 , r i b o f l a v i n e

calcium pantothenate

5, p y r i d o x i n e 0 . 5 , n i a c i n a m i d e 10, b i o t i n

0.05,

c y a n o c o b a l a m i n e 0 . 0 1 , v i t a m i n A a c e t a t e 4 a n d v i t a m i n E 10. E a c h r a t w a s given, intraperitoneally,

5 ¿iCi of g u a n i n e 2 - ^ C or g u a n i n e 8 - ^ C

for t h r e e c o n s e c u t i v e d a y s . T h e u r i n e w a s c o l l e c t e d d a i l y into

daily

flasks

1,

275 containing 0.25 ml of formic acid for five days following the initial in14 jection of guanine -

C. These procedures were performed in a sterile lam-

inar flow room. Materials, including food, water and cages, were sterilized by autoclaving. The guanine solutions were sterilized by millipore filtration. The combined urine from each group of animals was analyzed for the incorporation of radioactivity into biopterin by the procedure shown in Figure 1. The purified biopterin containing fractions from the animals RAT URINE (25 ml, 2-3 « CONCENTRATED) I DOWEX 1 » » ( H C 0 2 ® - F O R M , 45 ml VOL) • 0-30 ml H j O

. 0-450 m i l 5 N H C O j H

30-650mlH]0

I

BIO R I X 70 ( H ® — FORM, 2 6 m l VOL) ,

—

0-45mlH2O

•

45-360 m I H 2 O

0-220 ml IN H C O 2 H

It

SEPHAPEX G 2 5 {30 ml VOL)

,

1 O-llmlHjO

.

1 11-24 m I H 2 O

#

.

10%

1 24-60 m I H 2 0

'

I

SYSTEM A Rf=

008

014

020

030

I

90«

SYSTEM B

0 42

060

010

018

0 24

033

0 39

045

064

0 74

. I 470,000 CPM/uMOLE )| SYSTEM C

600,000 CPM/jtMOLE Rf=

0 35

0 60

114,000 C P M / p M O L E

T

SYSTEM A Rp =

0 24

112,000 CPM/|iMOLf

T

K M n O 4 -OXIDATION I SYSTEM C Rf= FI 8 1

0 41

ISOLATION OF RADIOACTIVE BIOPTERIN F R O M RAT URINE AFTER I P INJECTION OF GUANOSINE 2 - , 4 C

3550 CPM/fjMOLE

" T SYSTEM B Rf=

¡^¡4

3550 CPM/pMOLE

T

SYSTEM A Rf=

009

9S0 C P M / p M O l E

~r

SYSTEM F Rf =

0 75

T

SYSTEM Rf =

0 53

950 CPM/pMOLE D 950 CPM/jtMOl£

277 the loss of carbon 8 from the purine.

Kraut et al. (10) on the basis of the Crithidia bioassay reported that normal rat food contained a biopterin like compound. For this reason w e felt that the incorporation of radioactivity from guanine into pterins might be increased if the rats were maintained on a biopterin free diet. The rats maintained on the normal purina lab chow diet excreted about 20 ug of biopterin per day, the animals fed the synthetic diet excreted about 15 ug per day. Surprisingly, the incorporation of label from guanine 2 - ^ C into the isolated pterin-6-carboxylic acid was higher in those rats fed purina lab chow than those maintained on the synthetic diet.

Since glycine is a purine precursor w h o s e carbons become

incorporated

into positions A and 5 of the purine ring it should b e possible to demonstrate incorporation of radioactivity from glycine into urinary biopterin. Three germ free male rats, 7 weeks of age, maintained on purina lab chow, 14 were each administered, intraperitoneally, 20 ,uCi of glycine 2 -

C three

times daily for two consecutive days. The experiment was performed under sterile conditions as previously described. The urine was collected for 72 hours following the initial administration of glycine 2 - ^ C . A radioactive biopterin containing fraction was isolated from the urine as described in Figure 2 and then oxidized w i t h KMnO^ to yield pterin-6-carboxylic acid. The major portion of the radioactivity in the biopterin fraction remained associated w i t h pterin-6-carboxylic acid after the oxidation and the specific radioactivity of this pterin remained constant upon further purification.

2?8

(13 ml; 4x CONCENTRATED)

I

DOWEX 1 » g ( H C 0 2 © - F 0 R M ; 4 0 ml V O L )

T

I 0 - 4 0 ml H 2 0

4 0 - 6 0 0 ml H 2 0

0 - 4 5 0 ml 1.5 N

HC02H

I

B I O REX 70 (H®—FORM; 32 ml VOL)

t

I 0-70 ml H 2 0

I 70-200 ml H 2 0 I

Rp =

012

0.17 0.22

030

0.36 J

Rf=

0.03

0.05

I 0-200 ml IN H C 0 2 H

80,000 C P M / ^ M O I C 0.48

0.61

14,500 CPM/pMOLE

0.20 I

13,000 CPM/j*MOLE

SYSTEM C Rf=

0 29

0 58 J

13,000 CPM//»M01f

KMn04-OXIDATION

I

SYSTEM B I

9500 CPM/>tMOLE

SYSTEM A RF= 0.09 I

9200 CPM/^MOLE

SYSTEM C Rf= 0 41 9700 CPM/jiMOLE Fig. 2:

ISOLATION OF R A D I O A C T I V E BIOPTERIN F R O M RAT URINE AFTER

IP. INJECTION OF GLYCINE

2-UC

It was also possible to demonstrate pterin synthesis from guanosine in intact rat brain. Four germ free rats w e r e each administered,

intracister-

14 nally, 30 uCi of guanosine 2 -

C and killed 18 hours later. The brains

w e r e extracted w i t h 5 volumes of 5% TCA. To the combined supernatants 120 ug of carrier tetrahydrobiopterin w a s added w h i c h was followed b y iodine and KMnO^ oxidation (11) to yield pterin-6-carboxylic acid. F o l l o w ing purification of this compound it was possible to demonstrate r a d i o -

v X

P

307 phenylalanine is sigmoidal with an apparent K^ of around 0.1 mM. Although such curves for Phenylalanine hydroxylase have appeared in the literature [3,5,6] their significance in terms of enzyme mechanism have not been previously discussed. Sigmoidal Z^ curves are usually associated with polymeric enzymes. The two most frequently postulated mechanisms for sigmoidal kinetics are either (i) there is em interaction between catalytic sites, or (ii) the substrate is acting also as a modifier (effector) at a site other than the catalytic site, (i.e. the substrate acts at a regulatory, or allosteric site) to activate the catalytic site. For either mechanism the interaction between sites may involve either (i) a conformational change of the enzyme, or (ii) polymerization or depolymerization[7]. Hyperbolic kinetics, on the other hand, oocur either (i) when there is only one active site per enzyme molecule, or (ii) when there is more than one site, but each site acts independently of all others. The observation of sigmoidal kinetics with one cofactor (Pig. 1 A) but not with another (Fig.l C), although not excluded by these models, was not predictable. In the following experimentsother anomalous behavior of the enzyme became apparent. A model for the mechanism of regulation of phenylalanine hydroxylase activity is proposed which is consistent with these properties of the enzyme.

RESULTS Time Course of the Reaction.

The reaction velocity as a

function of time varies markedly depending on which cofactor is used and whether or not phenylalanine is present in the reaction mixture during temperature equilibration (Fig.2). Under standard assay conditions, in which the reaction

309 with tetrahydrobiopterin at the start of the reaction, the initial rate is very slow, and remains slow (Fig. 2 A). From these results it appears that the enzyme has two states, a low activity and a high activity state, and that interconversion from a low activity to a high activity state is effected by the substrate phenylalanine. This conversion can occur whether or not 6,7-dimethyltetrahydropterin is present. Conversly, tetrahydrobiopterin, if present, inhibits the initial activation by phenylalanine or, if enzyme is already activated, affects the equilibrium between the two forms of the enzyme.

Basis of the Sigmoidal K^ Curve

for Phenylalanine.

With

tetrahydrobiopterin as cofactor, the low activity of phenylalanine hydroxylase, if not preincubated with phenylalanine, suggested that the sigmoidal K curve for m phenylalanine could be a reflection of the extent to which enzyme is activated. Alternatively, enzyme may be fully activated, even at lower concentrations of phenylalanine, and the sigmoidicity may be produced by some other mechanism. To make this .distinction , enzyme was preincubated for 5 min with concentrations of phenylalanine varying over the range used for measuring the K curve.Reactions were then m initiated by addition of tetrahydrobiopterin and sufficient phenylalanine to bring the final concentration to a constant level in all reactions. The results of such an experiment are given in Fig. 3 and compared to a standard Km curve in which the phenylalanine concentration during the reaction is the same as that preincubated with enzyme. W h e n the rates were measured at a fixed high concentration of phenylalanine and plotted against the concentration of phenylalanine preincubated with enzyme, a sigmoidal curve was obtained similar to that of the standard Km curve. The displacement of the curve would be expected

considering that enzyme

which has been preincubated without phenylalanine still

312 there is good agreement b e t w e e n the apparent K m of p chlorophenylalanine as substrate a n d the c o n c e n t r a t i o n of p-chlorophenylalanine

that gives 50$ i n h i b i t i o n of the

r e a c t i o n w h e n phenylalanine is substrate. W h e n t e t r a h y d r o b i o p t e r i n is cofactor the K m and I^p differ by nearly two orders of m a g n i t u d e .

p - C h l o r o p h e n y l a l a n i n e as a n Activator of Phenylalanine Hydroxylase

The time course of the h y d r o x y l a t i o n of

p - c h l o r o p h e n y l a l a n i n e was m e a s u r e d w i t h either

6,7-dimethyl-

t e t r a h y d r o p t e r i n or t e t r a h y d r o b i o p t e r i n as cofactor. W i t h 6 , 7 - d i m e t h y l t e t r a h y d r o p t e r i n the rates are linear w i t h time, but there is a 1-2 m i n lag if enzyme is not

preincubated

in the presence of p - c h l o r o p h e n y l a l a n i n e . W i t h

tetrahydro-

b i o p t e r i n as cofactor and p - c h l o r o p h e n y l a l a n i n e as substrate the rate is non-linear and, if enzyme is not

preincubated

w i t h p - c h l o r o p h e n y l a l a n i n e , the initial rate is very

slow,

and remains slow. The ability of p - c h l o r o p h e n y l a l a n i n e

to

activate the enzyme therefore appears to be similar to that of phenylalanine

(cf. Pig. 2).

As noted above, under standard assay conditions

(i.e. all

r e a c t i o n components, except cofactor, temperature b r a t e d together) the i n h i b i t i o n of a r e a c t i o n , a fixed c o n c e n t r a t i o n of phenylalanine and

equili-

containing

tetrahydrobio-

pterin, increases w i t h increasing concentrations of p chlorophenylalanine

(Table 1). If the r e a c t i o n

components

are temperature e q u i l i b r a t e d separately, so that enzyme is not p r e i n c u b a t e d w i t h either phenylalanine or p - c h l o r o p h e n y l alanine the r e a c t i o n rates are m u c h lower but the same % inhibition is observed. If, h o w e v e r , enzyme is p r e i n c u b a t e d w i t h p - c h l o r o p h e n y l a l a n i n e and the r e a c t i o n initiated by simultaneous a d d i t i o n of phenylalanine and

tetrahydrobio-

p t e r i n there is i n h i b i t i o n of the r e a c t i o n at lower

concen-

trations of p - c h l o r o p h e n y l a l a n i n e , but as the p - c h l o r o p h e n y l -

314

Fig. 5. Effect of preincubation with substrate on (1) desensitized, (2), (3) native phenylalanine hydroxylase. (1) Desensitized enzyme preincubated or not preincubated with phenylalanine. (2) Native enzyme preincubated with phenylalanine (the same as Fig. 1A). (3) Native enzyme, not preincubated with phenylalanine. rate is produced (Fig.5 curve 3). A similar effect is observed when p-chlorophenylalanine is substrate. Desensitization removes the need for preincubation with p-chlorophenylalanine, converts the sigmoidal K m curves into a hyperbola, reduces the

value for p-chlorophenylalanine about

fifty-fold (Table 2), and has no effect on the V max.

Cofactor Tetrahydrobiopterin »

6,7-dimethyltetrahydropterin H

Substrate

Native Enzyme

Desensitized Enzyme KJH curve K ^ m M ) K^ curve K ^ m M )

phe p-Cl-phe phe

S S H

0.1 2.0 1.0

H H H

0.05 0.04 1.00

p-Cl-phe

S

2.2

H

1.00

Table 2. Effect of desensitization of phenylalanine hydroxylase on the reaction parameters. S: sigmoidal; H : hyperbolic. Experimental conditions as for Fig. 1.

315 With 6,7-dimethyltetrahydropterin as cofactor and phenylalanine as substrate the rates and the shapes of the KJH curves for phenylalanine are the same whether or not enzyme has been desensitized or preincubated with substrate. In all cases the

curves are hyperbolic. The only differences

are that if enzyme has not been either desensitized or preincubated with substrate a 1-2 min lag occurs (cf. Pig. 2B) before the maximum rate is attained. This lag also occurs when the substrate is p-chlorophenylalanine. On the contrary, when p-chlorophenylalanine is substrate, desensitization converts the otherwise sigmoidal

curve

to a hyperbola, and reduces the K m about three-fold (Table 2), without affecting the

V max.

DISCUSSION The preceding results can be interpreted in the following manner. The sigmoidal K m curve for phenylalanine (Fig. 1A), the dependence of activity on the conditions of preincubation (Pig. 2A), and the disparity between the substrate and inhibitory potency of p-chlorophenylalanine(Table 1, Pig. 4) when tetrahydrobiopterin is cofactor, and the changes that occur when 6,7-dimethyltetrahydropterin is used instead, all argue for the presence of two types of site for substrate, one catalytic and the other regulatory. This has been preposed previously [8]. The binding of phenylalanine, or related analogs, at this second site triggers some slow (1 2 minutes) change which increases the activity of the catalytic site. The fact that regulation can be eliminated without loss of enzyme activity (Fig. 5) shows that the second site has no catalytic function. The activation induced by the binding of phenylalanine at the second site is inhibited by cofactor, but only when this is tetrahydrobiopterin (Fig. 2A). Since the system must

317 ^

value (Pig. 1C, Table 2).

This indicates that the bind-

ing of phenylalanine at the regulatory site is not limiting for this reaction. High affinity of phenylalanine for the regulatory site is reflected in its much lower K m with native enzyme when tetrahydrobiopterin is cofactor. The

KJJJ

curves for cofactor are hyperbolic (Fig. 1B,L), with

the implication that cofactor binds only at the catalytic site. Phenylalanine hydroxylase activity, therefore, is controlled by substrate at a regulatory site, which is in turn modulated by binding of cofactor at the catalytic site. Prom our studies so far, it appears that in order to have this effect, cofactor must specifically possess a 6-dihydroxypropyl group, since the cofactor properties of 6-methyltetrahydropterin are identical to those of 6,7-dimethyltetrahydropterin [9]. Acknowledgements: Supported by USPHS Grants CA-15659, HD06576, and HD-04612. REFERENCES 1.

Kaufman, S., The structure of phenylalanine hydroxylase cofactor. Proc. Nat. Acad. Sci. U.S.A., 50 1085-1092 (1963) ~~

2.

Kaufman, S. and Levenberg, B., Further studies on the phenylalanine hydroxylas 6 cofactor. J. Biol. Chsin» 234» 2683 - 2688 (1959).

3.

Ayling, J. E., Boehm, G. R., Textor, S. C. and Pirson, R. A., Kinetics of Phenylalanine hydroxylase with analogs of tetrahydrobiopterin. Biochemistry 12, 2045 2051 (1973).

4.

Ayling, J. E., Pirson, R., Pirson, W. and Boehm, G. R., A specific kinetic assay for phenylalanine hydroxylase. Analytical Biochemistry 51, 80 - 90 (1973).

5.

Kaufman, S., The phenylalanine hydroxylating system from mammalian liver. Adv. Enzymology 3_5, 245 - 319 (1971).

6.

Ayling, J. E. and Helfand, G. D., Inhibition of phenylalanine hydroxylase by p-chlorophenylalanine; dependence

318

on cofactor structure. Biochem. Biophys. Res. Comm. 61, 360-366 (1974). ~~ 7.

Koshland, D. E., The molecular basis for enzyme regulation. The Enzymes, 3rd. edition, P. D. Boyer, ed., Academic Press, Vol. 1, 341-396 (1970).

8.

Tourian, A., Activation of phenylalanine hydroxylase by phenylalanine. Biochim. Biophys. Acta 242, 345-354 (1971)

9.

Ayling, J. E. and Helfand, G. D., Regulation of phenylalanine hydroxylase activity. Biochemistry (1975).

10. Ayling, J. E. et al. unpublished results 11. Prieden, C., Kinetic aspects of regulation of metabolic processes. The hysteretic enzyme concept. J. Biol. Chem. 245, 5788-5799 (1970). DISCUSSION Hemmerich: Is there a correlation between your substrate induced activation step in pteridine dependent oxygenation and the well-known coupling of oxygenation and pteridine oxido-reduction ? It would be interesting to know in your case, how tetrahydrobiopterin autoxidation is influenced by a) b)

the enzyme alone, the enzyme + p-Cl-phe under inhibiting conditions.

Is the rate of 02-consumption and the extent of H2O2 formation increased ? Ayling: a) There is no enzyme catalyzed oxidation of tetrahydrobiopterin in the absence of aromatic amino acid. b) When p-Cl-phe is used as an inhibitor of the enzymatic hydroxylation of phenylalanine with tetrahydrobiopterin as cofactor,

319 Poe: Have you measured the stoichiometry of ffy-biopterin and phenylalanine binding by the enzyme ? If there is an allosteric site for phenylalanine, there should be more binding sites for it than for the other substrate. Ayling: This would be difficult to do since phenylalanine does not bind tightly to the enzyme (see table 2). McCormack: Is there a substantial difference observed between inhibitory potencies of catecholamines (e.g. dopamine, norepinephrine) when dimethyltetrahydropterin and tetrahydrobiopterin are used as cofactors for phenylalanine hydroxylase ? Ayling: Dopamine, norepinephrine and epinephrine inhibit 3 - 5 times more with tetrahydrobiopterin as cofactor than with 6,7-dimethyl-tetrahydropterin. Bergmann: P-Chlorophenylalanine is a strong inhibitor of hydroxylation of tryptophan. Does it also inhibit hydroxylation of tyrosine to DOPA ? Ayling: According to Dr. Kaufman, p-chlorophenylalanine inhibits tyrosine hydroxylase too. Bergmann: Was the oxidation product of p-chlorophenylalanine identified chemically ? Ayling: Yes. It was shown several years ago by Guroff et al. at NIH that the main product of the hydroxylation of p-chlorophenylalanine by phenylalanine hydroxylase is m-chlorotyrosine, and a small amount of tyrosine. We have confirmed that this is also the case for the reactions which we have measured. Kaufman: There is a discrepancy between the Km values for tetrahydrobiopterin of phenylalanine hydroxylase reported by Dr. Ayling and by us. She has reported a value of about 22 uM whereas we have reported a value of 3 to 4 yM. Is there any explanation for these differences ? Ayling: All of our work has been done with enzyme in the phenylalanine-activated state. This is essential, since with 6,7-dimethyltetrahydropterin as cofactor the initial steady state velocity is zero unless enzyme is preincubated with phenylalanine (see Fig. 2B), or is desensitized. With tetrahydrobiopterin as cofactor, unless enzyme is preincubated with phenylalanine (see Fig. 2A) or desensitized (see Fig. 5), the rates which can be obtained are so low that it is difficult to make an accurate measurement of the Km.

322 principal

reaction for this is still the subject of some speculation

When grown under photoautotrophic conditions Euglena

graailis

[2].

contains

glycollate pathway enzymes and detailed studies of C11*!^ fixation show that the pathway is functional during photosynthesis of the pathway

[3"7].

The

in providing precursors for secondary metabolism

importance is shown

by a coincidence of maximal glycollate oxidation and maximal rates of DNA synthesis in division synchronized cells [6,8]. the glycollate pathway in Euglena

Flow of carbon

through

is controlled by the activity of key

enzymes particularly glycollate dehydrogenase whose synthesis is repressed by culture in %% C0 2

in air

[6].

In earlier work from this laboratory

[9] w e reported a net synthesis of

formyl and methyl derivatives of Hi,PteGlu as division synchronized cultures received light.

Euglena

As this synthesis was accompanied by increase

in

the levels of key enzymes of C-l metabolism, we concluded that a rapid turnover of C-l units took place in the light at a stage of the cell when high rates of DNA, RNA and protein synthesis occur.

cycle

Considering the

operation of the glycollate pathway in this species it follows that C-l units in the formyl- and methy l-Hi,PteGl u pools may arise by reactions closely related to the metabolism of recently fixed CO2.

When the present

studies were initiated such relationships remained to be examined. The present paper reports evidence that C-l units in Euglena formyl

arise at the

level of oxidation via the 10-HC0-Hi,PteGl u synthetase reaction.

A

close relationship between this enzyme of C-l metabolism and glycollate dehydrogenase suggested that such formyl groups are derived from metabolism of glycollate produced in photosynthesis. METHODS Euglena

gracilis

Klebs (strain Z) ATCC 12716 was cultured synchronously in

mineral salts medium [ 9 ] .

Cultures were maintained

in a l^rlO hr light:

dark cycle at 25°C and were aerated with sterile air (0.03% C0 2 during growth.

in air)

Cell divisions were restricted to the dark phase and

resulted in a doubling of cell numbers before the following light period. In experiments designed to repress synthesis of glycollate dehydrogenase, cultures were aerated with sterile 5% CO2 in air. alter synchrony.

This treatment did not

324 control cells.

In contrast high C0 2 treatment elevated the level of serine

hydroxymethy1 transferase.

The hydroxysu1phonate, added 10 hrs prior to

harvesting, reduced the activities of all three enzymes.

When these data

were expressed on a per cell basis it was clear that high C0 2 -treated cells contained much less total glycol late dehydrogenase and 10-HC0-Hi,PteGlu synthetase activity but five times the serine hydroxymethyltransferase activity of air grown cells. The effects of high CO2 treatment on these enzymes were readily reversed on transfer to air (Table 2). TABLE 2.

As the level of these enzymes is known to

ENZYME ACTIVITIES AFTER TRANSFER FROM HIGH TO LOW CO,

Hours of Che 4th cell cycle (light phase) 0 2 8 12

Enzyme Glycollate dehydrogenase (unlts/IO' cells) transferred to air remaining In 5% CO,

10

10 II

50 30

60 29

53

50 60

51 88

53 106

Serine hydroxymethyltransferase (mimóle product/hr/IO* cells) transferred to air remaining In C0 2 10-HC0-Hi,PteG!u synthetase (miimole product/hr/IO* cells) transferred to air remaining In 5* C0 2

n.d.

0.03 0.01

0.65 0.03

0.58 0.03

Cells were cultured Initially for 3 cell cycles In $% C0 2 In air then transferred to low C0j (air) at the start of the 4th cell cycle. Control cultures received the 5* CO2 treatment throughout, n.d. - not detected.

change during the cell cycle [6,9] cells remaining in 5% C0 2 as controls.

Analysis of Euglena

formate from C-2 of glyoxylate.

in air served

extracts revealed ability to produce The activity of this decarboxylase was

not however altered by high C0 2 treatment. Besides affecting enzyme activity high C0 2 and a-HPMS treatment also caused dramatic depletion of 10-HCO-Hi,PteG 1 uj_ 2 pool size (Table 3). effect involved both unconjugated and polyglutamyl derivatives.

This

In

contrast, unconjugated and polyglutamates of methyl Hi,PteG)u were substantially increased by this treatment so that total folate concentration of the cells was not appreciably altered.

In the presence of a-HPMS

both classes of folates were decreased with greatest depletion being noted

327 From the data presented w e conclude that glycol late metabolism in involves production of formate which can act as a source of via the corresponding synthetase reaction. ate oxidation, i.e.

Euglena

1O-HCO-hUPteGlu

Conditions which block glycoll-

inhibition by hydroxysu1phonates or repression of

enzyme synthesis, have a direct effect on production of C-1 units at this level of oxidation.

It is not clear, however, whether

10-HC0-Hi,PteGlu

synthetase is controlled by C0 2 concentration directly or whether the observed effect on this enzyme (Tables 1 and 2) is more closely related to endogenous changes

in formate pool size.

When synthesis of formyl folates is curtailed, the serine hydroxymethyltransferase reaction may become the principal biosynthesis.

route for C-1 unit

At high C0 2 concentrations the carboxylation of ribulose

diphosphate would be favoured rather than synthesis of phosphoglycolate [14].

Under such conditions of increased CO2 fixation, the cells require-

ments for serine and C-1 units might be met by metabolism of C3 iates of photosynthesis.

intermed-

These possibilities are the subject of our

present studies. Acknowledgement: National

This

investigation was supported by grants from the

Research Council of Canada to E.A.C.

REFERENCES 1.

Tolbert, N.E.: Glycolate pathway. In Photosynthetic Mechanisms in Green Plants. Nat. Acad. Sci. National Res. Council Publ No. 1145. Washington, D.C. pp 648-662 (1963).

2.

Tolbert, N.E.: Microbodies - Peroxisomes and glyoxysomes. Plant Physiol. 22_, 45-74 (1971).

3.

Lord, J.H., and Merrett, M.J.: The intracellular localization of glycolate oxidoreductase in Euglena gracilis. Biochem. J. 124, 27^281 (1971).

4.

Graves, L.B., Trelease, R.N., and Becker, W.M. : Particulate nature of glycolate dehydrogenase in Euglena: possible location in microbodies. Biochem. Biophys. Res. Comm. 44, 280-286 (1971).

5.

Codd, G.A., and Merrett, M.J.: Photosynthetic products of division synchronized cultures of Euglena. Plant Physiol. 47., 635-639 (1971).

6.

Codd, G.A., and Merrett, M.J.: The regulation of glycolate metabolism in division synchronized cultures of Euglena. Plant Physiol. 47., 640643 (1971).

7.

Murray, D.R., Giovanelli, J., and Smillie, R.M.: Photometabolism of glycollate by Euglena gracilis. Aust. J. Biol. Sci. 24, 23-33 (1971).

Ann. Rev.

329 DISCUSSION Elstner: Do you have an idea what the endogenous electron acceptor could be in the algal system of glycolate dehydrogenase ? Cossins: As far as I am aware, this is not known for any algal species to date. In our assay we employed 2,6-dichlorophenolindophenol as electron acceptor. Iwai: I think, the main metabolic pathway of glycine in plants and protozoa will be catalyzed by glycine decarboxylase rather than serine hydroxymethyltransferase. Do you have any information on the effect of CO2 concentration on the glycine decarboxylase ? Cossins: We have not investigated this to date. It is conceivable that CO2 concentration, by reducing the flow of glycollate carbon, would also affect glycine pool size. We plan to investigate some of these possibilities in the near future. Plaut: What is the distribution of the glyoxylate and glycine decarboxylase enzymes ? Cossins: Zelitch has shown in higher plants that the glyoxylate oxidase is in chloroplasts. Glycine decarboxylase is a mitochondrial enzyme. Huennekens: As a variation on the pathway shown in slide 1, is it possible that glyoxylate condenses directly with tetrahydrofolate and that the resulting adduct decarboxylates oxidatively to yield CO2 and 5,1O-methenyltetrahydrofolate ? Cossins: Theoretically this appears possible but there is no direct evidence that this occurs in Euglena. The rapid incorporation of formate carbon into adenine and the fi-carbon of serine plus the relatively high levels of 10-formyltetrahydrofolate synthetase would argue for the involvement of formate in the C-1 metabolism of Euglena. Elstner: Is your glyoxylate 'oxidase' C^-dependent - and how in your sys tem is oxygen activated ? Cossins: Our assays were carried out under aerobic conditions, with illumination and in the presence of excess catalase. We have not examined, in detail, the requirements of the reaction as the enzyme has been assayed in crude cell-free extracts.

Folate Derivatives and Patterns of C-1 Metabolism in Neurospora Crassa, Wild Type, Ser-1 and Formate Mutants G. Combepine, E.A. Cossins and P.Y. Chan

INTRODUCTION There is considerable evidence that glycine can act as a source of 5, IO-methylenetetrahydrofolate (5,10-CH2-Hi»PteGlu"'!) .

For example, the

a-carbon of glycine is readily incorporated into the 8-carbon of serine in bacteria [l], Saeeharomyees plants [5]. Peptoaooous

[2], avian liver [3], rat liver fj] and higher

Glycine decarboxylase, an en2yme extensively studied in glyoinophilus

[6,7] but also present in a wide variety of other

species [A,8] mediates this C-1 unit synthesis which could clearly have importance in synthesis of a number of folate-related cellular constituents [9]. In earlier studies of Neurospora

arassa

[10] we found that culture in the

presence of exogenous glycine was accompanied by elevation of serine hydroxymethyltransferase. aes oerevisiae

[11].

Similar data have been reported for

Sacaharomy-

The effect of this addition could clearly be related

to a possible production of 5,1O-Chh-HUPteG 1 u from glycine which could in turn alter the pattern of C-1 metabolism and stimulate production of methionine, serine and purines. The present studies have therefore examined this effect of glycine in more detail using the wild type of N. arassa and two mutants which have relatively low levels of serine hydroxymethyltransferase. -'The abbreviations used for derivatives of folic acid are those suggested by the IUPAC-IUB Commission as listed in the Biochemical Journal 102, 15 (1967): e.g. 10-HCO-Hi,PteG 1 u = N 1 0 -formy 1 tetrahydropteroyImonog 1 utamate.

333

i'[fÌWì?SgWr'i1ìi 60 80 FRACTION N U M B E R

120

Fig. 1. Separation of folate derivatives in extracts of N. crassa after y-glutamyl carboxypeptidase treatment. Extracts of (A) wild type; (B) Ser1 mutant; and (C) formate mutant were prepared after 22 hr growth at 25°C. Peaks were identified as: (a) 1O-HCO-H^PteG1u; (b) 1O-HCO-H^PteG1u 2 ; (c) 5-HCO-H i^PteG 1 u ; (d) 5-CH3 -H„PteGl u ; (e) H^PteGlu; (f) 5-HCO-Hi.PteGl u 2 and Hi,PteGlu2; (g) 5-CH3-Hi,PteGlu2; (h) and (i) unidentified polyglutamyl derivatives. Growth of L. aasei (•) and P. aerevisiae (o). When the wild type and C-24 were cultured in the presence of 1 mM glycine (Table 1) changes in the folate pool occurred.

In the wild type, folate

concentration rose 3"fold largely due to increased synthesis of Hi,PteGlu.

33^ This may however reflect synthesis of 5,'O-CHz-H^PteGlu which would appear as Hi»PteGl u under the conditions employed in this study. did not increase total folate concentration.

In C-2't glycine

However in the presence of

this supplement a peak of 5-CH3-Hi»PteGlu was detected.

Glycine also

increased peak f (Table 1) which Judging by its position and ability to support growth of both assay bacteria probably contains 5-HCO-HuPteGIU2 and HnPteGlu 2 . TABLE I .

THE EFFECT OF EXOGENOUS GLYCINE ON INDIVIDUAL FOLATE DFRl VAT IVES IN M. CliAdHA WILD TYPE, 5CT-1 Aim FORMATE MUTANT WILD TYPE

DERIVATIVES

MINIMAL K D I W

FORMATE MUTANT

MINIMAL fEDIlM + 1 >H GLYCINE

MINIMAL PEDUM

MINIMAL MED11H + 1 KM GLYCINE

A. 10-HCO-H.P1EGLU

6.20

2.60

9.40

2.00

B. LO-HCO-FUPTEGUJI

1.30

n.d.

2.50

0.25

C. 5-HCO-H.PTEGLU

7.85

1.90

1.00

0.45

D. 5-CH,-H»PTEGLU

6.25

n.d.

n.d.

1.25

E. H.PTEGUJ

1.30

61.50

5.00

7.25

AND H»PTCGLUI

1.20

n.d.

1.35

9.40

G. 5-£H,-IUPTEGUJI

1.60

10.60

n.d.

n.d.

8.5

27.0

8.00

7.50

19.25

20.60

F , 5-HCO-HT.PTEGUUI

TOTALS: BEFORE HYDROLYSIS AFTER HTCROLYSIS

25.7

75.6

N.D. - NOT UblbClbl) EXPRESSED AS Ug/G. DRV WT. The levels of four key enzymes of C-l metabolism were also compared (Fig. 2) in the wild type and mutants.

The Ser-1 mutant contained all four

enzymes at levels comparable to the wild type, the only exception being the lower levels of serine hydroxymethyltransferase.

In the formate mutant

10-HC0-hUPteGlu synthetase, 5,10-CH 2 -H^PteGlu dehydrogenase and 5,10-CH 2 Hi,PteGlu reductase activities were above those of the wild type. contrast serine hydroxymethyltransferase was difficult to detect.

In This

finding is no doubt related to the ability of C-2k to utilize exogenous formate and inability to growth when only qlycine or serine are supplied. The effect of growth in 1 mM glycine on these enzyme levels is also shown in Fig. 2.

With the exception of 5,10-CH2-Hi»PteGlu dehydrogenase in 0-2^,

this supplement increased specific enzyme activities.

These data suggest

335 4.8

o E c

o v. a

2.4

O) \E

1.2

_œ O E c

*/t

j)

c Ê

0

d.

c.

c E 180 •s O k.

750

a

a> 500 E •Î E

c E s

120 a> E 60

250

o E

a WILD TYPE

SER1

r-sa FORM- WILD 9 TYPE

SER • 1

FORM9

F i g . 2. L e v e l s of key enzymes of C-l metabolism in N. orassa w i l d t y p e , Ser-1 and formate mutants. E x t r a c t s were prepared a f t e r 22 hr growth in media supplemented w i t h 1 mM g l y c i n e ( Q ) ; or lacking t h i s supplement ( • ) . (a) 5,10-CH2-Hi»PteGl u dehydrogenase; (b) 1 0-HC0-Hi,PteGl u s y n t h e t a s e ; (c) s e r i n e hydroxymethyl t r a n s f e r a s e ; (d) 5 ( 10-CH 2 -Hi t PteGlu reductase. that g l y c i n e or a product d e r i v e d from t h i s amino a c i d a f f e c t e d and turnover of C-l u n i t s w i t h i n the f o l a t e pool. ance (see I n t r o d u c t i o n ) the f i n d i n g bacteria,

synthesis

Considering the

import-

of g l y c i n e in generation of 5,10-CH2-Hi

3^6 Table III.

Summary of the purification of the dlhydrofolate synthetase

from S. indiaa. Fraction fraction

Total

protein mg

I. Extracts II. Am^SO^ ppt.

Tota1

activity

Specific activity

units

54 ,310

196,000

units /mg 3.6

Purification

Y.i e.l d

ratio 1.0

X 100

22 ,140

124,500

5.6

1.6

63.5

III. 1st DEAE-Sephadex column

2 ,592

56,370

21.7

6.1

28.8

IV. 2nd DEAE-Sephadex column

688

33,800

49.3

13.8

17.2

V. 1st Sephadex G-200 column

63

23,590

374.4

103.7

12.0

VI. 2nd Sephadex G-200 column

45

18,270

406.0

112.5

9.3

31

14,448

466.0

129.1

7.4

VII. DEAE-cellulose column

One unit of enzyme activity was defined as 0.1 mpmole of folic acid equivalent formed for 30 minutes under standard assay conditions!!!].

Time (minutes) Fig. 1.

Sedimentation patterns of the dlhydrofolate synthetase from

S. indioa. The solution contained 0.42 % of the enzyme In 0.01 M Tris buffer at pH 7.5 containing 0.1 M KC1 and 10 mM of 2-mercaptoethanol. Photographs were taken at the indicated times after reaching 60,000 rpm at 5°C. stoichiometry of the reaction is discussed below. After the reaction 14 32 C or ATP-y- Pi, radioactive nucleotides were separated on

using ATP-U-

a Dowex I X2 (formate form) column and their quantities were determined 32 from the radioactivity. The amount of Pi formed was determined by the following:

Equal volumes of 2 % sodium molybdate and 1.5 N sulfuric

acid and four volumes of isobutanol were added to an aliquot of the reaction mixture and the whole was shaken vigorously for 10 seconds.

After

letting the mixture stand for 1 minute, the radioactivity of an aliquot of the upper organic solution was measured with a liquid scintillation 2+ spectrometer. The reference experiment was performed without Mg . When

347 dihydropteroic acid was left out of the reaction, no ADP was detected. 14 Products formed enzymatically under standard conditions with ATP-U- C were ADP-U-

C and dihydrofolate, but no AMP was detected.

When ATP-y-

PI was used as the substrate under standard conditions, dihydrofolate and

PI were detected (Table IV).

Table IV.

We estimated that each mole of ADP

Stoichiometry of the reaction products Products (mu mole)

Substrate

H,. Folate

ADP

2.7

3.1

ATP--u- 14 c ATP--Y- 32 Pi

-•

Pi

2.3

2.5

The reaction was carried out in an argon atmosphere. and Pi was formed from one mole of ATP, and that one mole of dihydrofolate was simultaneously formed by the dihydrofolate synthetase reaction.

The

dihydrofolate synthetase from S. indioa required a divalent cation and a univalent cation for full activity[12]. was satisfied by Mg 2+ with 5 m M of Mg on its kinetics. +

2+

, Mn

2+

2+

or Fe

The divalent cation requirement

, and maximum activity was obtained

. Mg-ATP appears to be the required substrate based The univalent cation requirement was satisfied by K + ,

+

NHH , or Rb , and maximum activity was obtained with about 100 mM of each. K (10 mM) produced 70 % of the maximum activity. partly replaced by T l

+

+

and Cs .

Na

+

and L i

+

The effect of K

were ineffective.

was

A corre-

lation seems to exist between the ionic radii of the effective cations and their abilities to activate the enzyme.

To study the effect of K + ,

the kinetic constants of the substrates for the reaction (ATP, dihydropteroate or L-glutamate) were determined at concentrations of 7-5 and 100 mM of K + .

Prom the results shown In Table V, an Increase in the K +

concentration decreased the Km values for dihydropteroate and L-glutamate, with no effect on ATP, and It increased the Vmax for ATP and dihydropteroate.

The conformational change of protein in the presence or absence of

univalent cations has been widely studied[12]. univalent cations, e.g. the K

+

In the absence of

ion, fomyltetrahydrofolate synthetase

dissociated Into four subunits.

The S value of the dihydrofolate synthe-

tase in S. indioa, however, showed no change on ultracentrifugal analysis in the presence or absence of K + .

These results suggest that K + may

3^8

affect the binding of dihydropteroate and L-glutamate to the enzyme. Table V.

Effects of the K + concentration on the kinetic constants of Substrate ATP Di hydropteroate L-Glutamate

K+ Cone. mM 100 * 7.5 100 * 7.5 100 * 7.5

Km M 2.2xl0"4 2.9xl0"4 2.5x10"' 1.3x10"® 2.5x10"' 9.1x10"3

Relative V 1.0 0.85 1.0 0.63 1.0 0.97

Activity was measured under standard assay conditions except that the synthetase was dialyzed for 48 hours against 10 mM Tris buffer, pH 8.0, to which 50 mM 2-mercaptoethanol had been added. * Activity was measured under the above conditions with 7•5 mM of K . Homopteroic acid is an intermediate in the synthesis of homofolic acid and occurs as a contaminant in certain commercial preparations of homofolic acid.

Kisliuk et al reported that tetrahydrohomopteroate displayed

an inhibition against a pyrimethamine-resistant strain of the Plasmodium aynomolgi of a monkey parasite[13].

The growth of Lactobacillus oasei

was completely inhibited by 1 yM of homofolate or 40 yM of hopiopteroate. As shown in Table VI the dihydrofolate synthetase of S. indiaa was Table VI. Inhibition of the dihydrofolate synthetase from S. indioa by homopteroate and its reduced compounds. Additions Homopteroate

Dihydrohomopteroate

Tetrahydrohomopteroate

inhibited

wM 0 10 100 0.1 1 5 10 50 100 5 10 SO 100

Folate equivalent liunoles 198 210 205 192 172 158 139 86 50 201 192 172 130

Inhibition I 0 0 0 3.1 13.3 20.4 29.8 56.7 74.8 0 3.0 13.3 35.4

by the addition of reduced forms of homopteroic acid.

A

stronger inhibition by dihydrohomopteroate was observed than by tetrahydrohomopteroate.

This suggests that the inhibition by tetrahydrohomo-

pteroate in vivo and -in vitro may result from oxidation of the tetrahydro form to the dihydro form and that the true inhibitor of the synthetase

3^9 may be a dihydrohomopteroate.

Other properties of the synthetase are

surrmarized In Table VII. Table VII. Properties of the dihydrofolate synthetase purified from S. indiaa. Sedimentation c o e f f i c i e n t

3.9 S

Molecular weight

47,000

Stable pH

8

Optimum pH

9.0

Km values f o r dihydropteroate

1.3 uM 250 yM

L-glutamate ATP Activated by M g

290 yM 2

Wt

Fe 2 t K + , NH^, Rb+

Inhibited by p-chloromercuribenzoate

GTP cyclohydrolase The elimination reaction of formic acid from GTP is catalyzed by an enzyme, called GTP cyclohydrolase, dihydroneopterin triphosphate synthetase or GTP-8-foimylhydrolase, which has been partially purified from E. aoli [14], L. plantarum[3], Comamonas sp.[5] and Streptomyoes xnmosus[15], respectively.

The properties and reaction products of the

enzyme from each source differed. We found that S. indiaa produced considerable amounts of pteridlne compounds (L-t/zreo-neopterin and isoxanthopterin) and that extracts of S. indiaa catalyzed the formation of fluorescent compounds from GTP. The purification and characterization of the GTP cyclohydrolase from S. indiaa were investigated.

Growth

was allowed to proceed for 18 hours at 37°C under aeration. The frozen cells (about 1.8 Kg) were ruptured by grinding them with an equal volume of sea sand for 2 hours at 4°C after thawing. The broken cells were suspended in 20 volumes of 10 mM Tris buffer, pH 7.6, containing 4 mM of 2-mercaptoethanol, and centrifuged. The supernatant was treated by ultrasonication for 30 minutes at 10°C, then centrifuged. The supernatant extract was used as the source of the enzyme.

A dialyzate of the

50 to 70 % ammonium sulfate fraction was purified by DEAE-cellulose and Sephadex G-200 column chromatographies.

Purification steps and yields of

the enzyme are summarized in Table VIII. The final enzyme preparation

350 Table VIII. S. indioa.

Summary of the purification of the GTP cyclohydrolase from Specific Activity

Fraction

pyrophosphorylmethyl dlhydropterin

hydroxymethyl dlhydropterin

units / pg of protein Crude extracts

6.04

1.43

Aflmonlum sulfate fractionation

6.48

2.73

1st DEAE-cellulose column

26.7

0.693

2nd DEAE-cellulose column

44.0

0.129

1st Sephadex G-100 column

100

2nd Sephadex G-100 column

185

1st DEAE-Sephadex A-50 column

460

2nd OEAE Sephadex A-50 column

527

not detectable

• •

One unit of enzyme activity was defined as one mpmole of formate eliminated for 40 minutes under standard assay conditions. was 286 fold purer in specific activity than the original extract.

GTP

cyclohydrolase activity was measured by the following two methods:

The

labeled formic acid eliminated frcm GTP-8-"^C was oxidized by mercuric acetate to "^COa, then the li+ C0 2 was trapped by a g-phenylethylaminemethanol solution on filter paper in vials.

A scintillator was added

and the solution was counted with a Packard Tri-Carb scintillation spectrometer.

The other method was fluorometric, using a Hitachi spectroflu-

orometer at activating (355 nm) and emitting (440 nm) wavelengths.

After

the reaction mixture had been boiled and centrifuged, the fluorescent intensity of the clear supernatant was measured. conditions:

Standard assay

Incubation mixtures contained 0.1 M Tris buffer, pH 8.6; 50

mM 2-mercaptoethanoi; 0.2 m M sodium formate; 0.2 mM G T P - S - ^ C (0.2 y d ) and the enzyme in a total volume of 0.1 ml.

Reactions were carried out

in small tubes connected with counting vials by rubber tubes.

Each vial

contained Toyo No. 2 filter paper (20 x 80 mm) moistened with 50 % f5phenylethylamlne In methanol.

Reactions were started by injecting the

enzyme and were stopped by adding 10 % TCA, then they were oxidized with 0.2 N acetate and 0.66 N mercuric acetate.

After incubation at 37°C for

2 hours, 10 m l of scintillator containing 2,5-diphenyloxazole(4 g) and l 3 4-bis-2-(5-phenyloxasolyl)benzene

(100 mg) in one liter of toluene was

added to each vial. Formate elimination was proportional to the fluorescence formation. Purified cyclohydrolase specifically utilized GTP as substrate.

Enzyme

351 activity was inhibited by nucleotides such as GDP and ATP, as shown in Fig. 2. The requirement for GTP was competitively inhibited by GDP as GMP

0

1

2

3 Cone.

4

5

6

(10^)

Fig. 2. Effects of nucleotides on the GTP cyclohydrolase from S. indiaa.

Fig. 3-

Effect of GDP on the GTP cyclohydrolase from S. indiaa.

shown in Fig. 32+

Enzyme activity was inhibited by metal ions as shown in

Table IX.

Ca

and monovalent cations had no effect.

Table IX.

Effects of metal ions on the GTP cyclohydrolase from S. -indiaa. Formate eliminated Metals Non« N.* K+ Rb+ Li* C.2*

Fe 2 + Co 2 * HI 2 * Cu 2 * Zn 2 * Cd 2 * «1*

(mitrale / ng of protein) 74 0 74.0 74.0 74.0 74.0 74.0 62.9 62 2 29.4 59 2 50 3 24 4 0 0 0 3.7

Inhibí tion t 0 0 0 0 0 0 15 16 60 20 32 67 700 100 100 95

Standard assay conditions without 2-mercaptoethanol were used. The Indicated metal ions were added in the form of chloride at 5 nW.

352 Other properties of the purified GTP cyclohydrolase are summarized in Table X. Table X.

Properties of the GTP cyclohydrolase frcm S. indiaa. Molecular weight

170,000

Optimum pH

8.2

Km value f o r GTP

3.7 yM I n h i b i t e d by Zn 2 t Cd 2 t Hg2t A l 3 t Cu2t Fe 2 t Ni2t Co 2 + 2+ 2+ 2Mg , Mn , p-chloromercuribenzoate

Although the pteridine product of the GTP cyclohydrolases in E. aoli and Comamonae sp. and the dihydroneopterin triphosphate synthetase in L. plantarwn have been indicated to be a dihydroneopterin triphosphate, the product in S. indiaa differed.

We present here some of our recent

findings relevant to the reaction product formed from GTP by the GTP cyclohydrolase in S. indiaa.

After many incubations, the product(X-

compound) was isolated and purified by DEAE-cellulose column chromatography and TLC-electrophoresis. Table XI.

As shown in Table XI, X-compound had

Phosphate contents of the X-compound and GTP Phosphate contents Co

"

lpounds

Observed

Theoretical

moles / mole

compound

Acid hydrolyzed X-compound

2.7

3.0

AP-treated X-compound

1.2

1.0

PD- and AP-treated X-compound--

1.7

2.0

GTP

2.6

3.0

*X-compound treated with alkaline phosphatase from calf intestine. **X-compound treated with phosphodiesterase from beef heart and with alkaline phosphatase. three phosphate radicals; one mole of phosphate was easily liberated by alkaline phosphatase and two moles of phosphates were liberated by phosphodiesterase and by alkaline phosphatase.

The absorption spectra

of the oxidized product, the X-compound are shown in Fig. 4.

The

excitation and emission spectra of X-compound are shown in Pig.5-

X-compoud

APase-treated

WAVELENGTH ( NM )

WAVELENGTH

Fig. 4.

Absorption spectra of X-compound.

Pig. 5-

Excitation and emission spectra of X-compound.

HAVE

L E N G T H

X-compoud

( NM )

( N H )

These results suggest that X-compound has a cyclic phosphate structure in its neopterin molecule, which differs from the neopterin cyclic phosphate isolated from a crude reaction mixture of S. indioa, and the neopterin triphosphate identified in other microorganisms.

Elution profiles of

X-compound and D-erz/tfo-o-neopterin triphosphate, prepared from GTP treated with the GTP cyclohydrolase of E. aoli, are shown in Fig. 6. X-compound

¿L Neopterin-PPP.

ui r

t• Fig. 6. Elution profiles on high pressure liquid chromatography of the X-compound and neopterin triphosphate. GTP was used as the internal standard. The Shimadzu type 830 high speed liquid chromatography was used.

354

H*COOH

H N W i t npound]

P|PPOH2Q

6TP

CNP- H 2 I X

GDP, ATP, SOlHeavy metal ions SH - reagents

\ -Pi

1 + Pi

0 HN HzN^N L-threo-H?NP /accumulated in the \ c u l t u r e medium

H 2 Pterin-CH 2 OH ATP

(accumulated in the ' V cui ture medium

m2+ Mg

AMP CHJOPP;

_„„„,.,„,,, l(accumulated in the"» Folote coenzymes J C(j1t ( j r e ^ m

HJN'

HJN-^-COOH




tu C

rH B

H a) m id o

01 G

I i

I 1

•a c 10 ui rH rH 10 O O) id H-t

M

m

m

m

m

LO

LO

m

m

^ u •0 *rH >1 n fi a) nj -M

co

r-H ro

O

M

m

rH

m ro

CN M

r-» r-

ITI CN

VO

O + O ai

o , O, +1 +1 co 10 CN

O, +1 rH

O, + | CO m

î i m

P 0

H —

[TJ

m P 3 G rH B

*

o

O

O

EN

(TI

ai

•—i

rH

•—i

CD

CO

ID

O

ó

O

M

•

•

— i 1 1

I 1

•

rH

•

I

1

«

CM

•

i—i

—•

O

O

O

•

CT)

01

O rH

•—1

r-H

1 1

I 1

1 1

^

là

—-

r-H

-—-

1

•—-

10

m

O

O

N CD

O

O m

o lO

O

o rH

CN

1 1

•

o in •—i

1

1

O

o co rH

C/!

a) CD G id G u >i X) •o

1 1

c •rH S T3 id -p IH id

O

^ CM

u M +1 G 10 a) B

pi 5

«3 CM —i

S

B

1—1

CN

[

1

o rH

S

Í I

+ IO

—-

rH

10 IO

—

co

+1 i in +1 +1 m IO p» m rH r-H i—i

CN

iH

m

rH

S

CN

H >

CM

r-l

—-

c -rH ÍH —

rH

IO

in

CD P

O

•

m -P — i 1 o id a) S 'i i s

CN

O CM

M

(0 C1J en -H TD AJ

I

ja

01 id

-ci cu ^ 3 en id cu B

rH

CN

^ 3 en id a) S

ro

0)

J >I

XI

T3 0 Id en en id

ro

378 tetrahydrofolic acid (II) (13) and 2 -

14

C-5-methyltetrahydrofolic

acid (I)

(14) a r e given orally to the rat the principal metabolite in the f i r s t 24 14 hour urine is 5-methyltetrahydrofolic acid (I). When 5 - C - 5 - m e t h y l tetrahydrofolic acid (I) (13) is administered orally to the rat although radioactive compounds are found in the f i r s t 24 hour urine none could be identified as folates.

Thus in the rat both 10-formyltetrahydrofolic acid

(II) and 5-methyltetrahydrofolic acid (I) a r e readily converted into 5 methyltetrahydrofolic acid (I) and the l a t t e r compound rapidly exchanges the N5-methyl group in biological r e a c t i o n s . S i m i l a r r e s u l t s a r e found in man when 10-formyltetrahydrofolic acid (II) and 5-methyltetrahydrofolic acid (I) a r e administered.

Both compounds

a r e rapidly metabolised into the folate pool of which 5-methyltetrahydrofolic acid (I) is the m a j o r component and 10-formyltetrahydrofolic acid (II) is the minor component (16).

The N5-methyl group of 5 - m e t h y l -

tetrahydrofolic acid (I) rapidly exchanges (17). Tetrahydrofolic acid (VII) is readily oxidised to dihydrofolic acid (VIII) at neutral or alkaline pH (18, 19).

10-Formyltetrahydrofolic acid (II)

will be oxidised at a s i m i l a r r a t e to give 10-fcrmyldihydrofolic acid (IX) which will be oxidised to 10-formylfolic acid (X) (20).

The l o s s of the

p-aminobenzoylglutamic acid side chain which o c c u r s on the subsequent oxidation at room temperature of dihydrofolic acid (VIII) to form xanthopterin (XI) is unique to dihydrofolic acid (VIII).

7, 8-Dihydropterins

such as 7, 8-dihydrobiopterin (XII), in which the C6 side chain cannot provide a nucleophilic migrating group a r e oxidised without l o s s of the side chain (18,19,21).

Thus after storage 10-formylfolic acid (X) should

be formed from the 10-formyltetrahydrofolic acid (II) present in foodstuffs.

The p r e s e n c e of 10-formylfolic acid (X) in foodstuffs has been

reported (22, 23) although it was not c l e a r l y established whether or not the 10-formylfolic acid (X) identified was originally present or was an analytical artifact.

379 COOH

c h

H N H,N

n h Q c o n h

2

h N

ch

_ C H

2

N H Q C O N H

HN

H2N

COOH ÇH ch

N

H

2

oxidation neutral or alkaline solutions

COOH

m

H2N

çh CH2

2

2

CH2

I

VITT

COOH

CHO

COOH

CH2N Q C O N H

ÇH

—

ch ch

N IX

2

I

I

2

2

COOH

oxidation

380 When 2 -

14

C - 1 0 - f o r m y l f o l i c acid (X) was given o r a l l y to r a t s no radio 14 active folates were found in the f i r s t 24 hour urine. A number of C-

labelled compounds (pterins?) were obtained, one of which was tentatively identified as 6-hydroxymethylpterin (XIII) (13).

Thus in the rat

10-formylfolic acid does not enter the folate metabolic pool at a m e a s u r able rate but instead is degraded to s i m p l e r pterins.

The failure of 10-

formylfolic acid (X) to enter the folate metabolic pool is in agreement with other observations that it is not reduced by dihydrofolate reductase (24).

S i m i l a r r e s u l t s were obtained in man (16).

Although o r a l 10-

formylfolic acid (X) passed the gut wall effectively it was not m e t a b o l ised and was e x c r e t e d unchanged in the urine.

As 1 0 - f o r m y l f o l i c acid

(X) is fully active for L . c a s e i in the standard microbiological a s s a y and is probably present in many foodstuffs the use of microbiological a s s a y to determine the availability of food folates for man is misleading. 5-Methyltetrahydrofolic acid (I) is oxidised by molecular oxygen at about one-tenth of the rate of tetrahydrofolic acid (due to s t e r i c hindrance about N5) (19,25) and f o r m s 5 - m e t h y l - 5 , 6 -dihydrofolic acid (XIV). The oxidation rate is i n c r e a s e d by increasing pH or by t r a c e s of cupric ions (25).

S i m i l a r but v e r y rapid oxidations occur with e x c e s s f e r r i c

ions, cupric ions or quinones (25, 26).

5 - M e t h y l - 5 , 6-dihydrofolic acid

(XIV) rapidly r e a r r a n g e s to 5 - m e t h y l - 5 , 8-dihydrofolic acid (XV) at pH values l e s s than 4 5 (25, 26) o r may add a molecule of water to give 4 a h y d r o x y - 5 - m e t h y l - 4 , 5, 6, 7-tetrahydrofolic acid (XVI) (25).

Thus a f t e r

a time foodstuffs may contain a mixture of (I), (XIV), (XV) and (XVI ). 5 - M e t h y l - 5 , 6 -dihydrofolic acid (XIV) is inactive for L . c a s e i when determined by an aseptic a s s a y technique in the absence of a s c o r b i c acid (26).

However, in the p r e s e n c e of a s c o r b a t e (XIV) is rapidly reduced

to 5-methyltetrahydrofolic acid (I) so that in the conventional analyses where e x c e s s ascorbate is used (XIV) appears to be fully active (26). Both 5 - m e t h y l - 5 , 8-dihydrofolic acid (XV) and 4 a - h y d r o x y - 5 - m e t h y l - 4 , 5 , 6 , 7-tetrahydrofolic acid (XVI) a r e inactive for L. c a s e i under all con-

381

CONH HN

COOH I CH I C H 2¿ I

H2N

C H ,2 I COOH

^N

XEL

CH 2 NH

H N H2N

^N

\ /

CONH

COOH I CH I CH,2 I CH,Z I COOH

XE

CH,

O

N

HN H 2 N' \

CH Z NH H

A

M

/ N'

H.

M

COOH I L J CONH CH I CH, I CH,2 I COOH

582 ditions.

When 5 - m e t h y l - 5 , 8-dihydrofolic acid (XV) and 4 a - h y d r o x y - 5 -

m e t h y l - 4 , 5 , 6 , 7-tetrahydrofolic acid (XVI) a r e orally administered to man there is no change in the s e r u m folate level (Table 2) (27).

That

this is due to failure of these compounds to enter the folate metabolic pool and not to failure to pass a c r o s s the gut wall is suggested by studies 14 in the rat in which o r a l 2 - C - 4 a - h y d r o x y - 5 - m e t h y l - 4 , 5, 6, 7 - t e t r a h y d r o folic acid (XVI) as the naturally occurring d i a s t e r e o i s o m e r is absorbed, distributed and e x c r e t e d

(14).

When 5 - m e t h y l - 5 , 6 -dihydro-

folic acid (XVI) is given orally to man there is a s m a l l quick r i s e in s e r um folate (Table 2) (2 7).

The s m a l l n e s s of this r i s e together with the

e a r l y maximum and the known acid lability suggested that this compound was r e a r r a n g e d to the inactive 5 - m e t h y l - 5 , 8-dihydrofolic acid (XV) in the stomach acid.

Only that fraction which avoided contact with stomach

acid would then be available for absorption.

Confirmation of this was

obtained by the simultaneous oral administration of sodium bicarbonate (to produce t e m p o r a r y achlorhydria) and 5 - m e t h y l - 5 , 6-dihydrofolic acid (XIV) to normal subjects when much l a r g e r serum folate r i s e s were obtained. (Table 2) (2 7).

P e r n i c i o u s anaemia patients have permanent

achlorhydria and again oral administration of 5 - m e t h y l - 5 , 6-dihydrofolic acid (XIV) gave much l a r g e r r i s e s in the serum folate (Table 2) (2 7). T h e s e observations suggest that that portion of food folate present as 5 m e t h y l - 5 , 6 -dihydrofolic acid (XIV) which would be measured as folate by the conventional L

c a s e i a s s a y is much l e s s utilisable by man than 5 -

methyl-tetrahydrofolic acid (I).

Previous workers have noted that p e r -

nicious anaemia patients have higher average levels of s e r u m folate than normal subjects (2 8).

This effect has been a s c r i b e d to the "methyl

folate t r a p " brought about by the reduction in B\2 l e v e l s .

It has never

been c l e a r why the reduced rate of loss of the methyl group should cause retention of 5-methyltetrahydrofolic acid (I) and other investigators had shown that there is no difference in the folate p l a s m a c l e a r a n c e r a t e s of normal subjects and s u b j e c t s with pernicious anaemia (29, 30).

It now

383

m 0 •H ni P O -rH o "rH E m C eu -r-i M ta 0) c ft ed 01

> l-f X T3 •H O m

•

-r-t rH 0 UH 0 M T3 >1 A •rH Tí 1 VO •• m i

CO g •p 3 o •rH a) •o •n 0 1 o 5 a S S A > I -ri X in fl ^ O IH O ^ T3 I >1 in a i m^ 5< -P H 0 m > M -P y. •d M — >i a A « 1 P -H nj a) o vr B nj ^ G eu o -P -H ihh -P ed 1 H

,1+3.

1—1 >1 e H 0 m 1 o rH

tu 3-

,

U < Q

o fa

ci CO

+1

>h g

01 3. ?l

w + 1 c m a) 0

386

io m rH

td LO

>i i

n

fö LT)

tj

m < B

1

i

fc

LT) •

S

1

CO rH

1

1

1

i

O X3 CO H

c •H

>i

TD

Q

- s Ü

0) -p 10 i—i o fe

'—'

.—.

•d 1

a

Xi

'—'

O

.—

LT)

•

—

»

rö

XI Vf r^ O

,—.

iH in

*—'

i

—'

O

O

.—.

1

^

LO CN s fö CO

XI

*—'

o

CM m

•—'

1

.—.

r-

•

i

o

w Xi

-—.

1

Xi

—'

.—. Xi

rö

1

•

O

—

U]

i

—'

CM

>1 X) a) p ro H

CN

—'

x: -p

m

1

Xi

Q)

M

•H

.—.

CN O

.—-p 10 Pi

¿

"fÜ LO

Òi H Cl) Ol CD P 10 — i1 0 4H a>

>

•H 4-1 O to 0 •H u (0 M

(0

-p •C Ol •rH 1 T3 O X! 0 rH •H a

n a) o< Ci 3Oì

C\I

rH

•a 0) a •H cfl -P eu n

MH 0

Oi 3-

^

eu en 0 •a rH (0 0

,, XI

X

\ Ol 3-

H IT) rs

•H U (0 Ü

rH Ol 0 •H 1 >1 TD x: 0 ifl XI n p a) 0 rH p •H rH y >i X! u p Q) Q) E 1 Ol m 3. si

ft

ai CN iH

UH 0 a) U) 0 •O rH (0 !H 0

„

01

—'

—

0 rH •H

o

08 •a

-—

,—.

ai

V c 10 0 -H •H ^

X Ol 3U"1 CD CN

387 folate showed no evidence of saturation.

F r o m these data it may be

concluded that folates in the rat exist in two distinct metabolic pools. The f i r s t pool c o m p r i s e s serum and presumably tissue fluid and cyto plasm and is in rapid exchange with the external environment by a b s o r p tion and e x c r e t i o n .

The second pool c o n s i s t s of t i s s u e retained folate

and only slowly exchanges with the folates of the f i r s t pool.

Studies of

urinary excretion and liver retention of radioactivity in the rat after •14 14 o r a l 2 - C - 1 0 - f o r m y l f o l i c acid (X), 2 C - 5 - f o r m y l t e t r a h y d r o f o l i c acid 14 14 OCVII), 2 - C - 5 , 1 0 - m e t h y l i d i n e tetrahydrofolic a c i d ( X V I I I i 2 - C - 1 0 14 formyltetrahydrofolic acid (II), 2 - C-5-methyltetrahydrofolic acid 14 (natural d i a s t e r e o i s o m e r ) (I) and 5 - C-5-methyltetrahydrofolic acid (I) all showed the same pattern as with folic acid (III). (Tables 3 and 4) (13). Thus the folates derived f r o m these compounds also exist in two distinct metabolic pools s i m i l a r to those derived from folic acid (III). O r a l administration in man of folic acid (III),

5-methyltetrahydrofolic

acid (I), 10-formylfolic acid (X), 10-formyltetrahydrofclie acid (II), 5 formyltetrahydrofolic acid(XVII)and 5,10-methylidinetetrahydrofolic acid (XVIII)gives a rapid r i s e in serum microbiologically active folate reaching a maximum in l|- - 2 hours and t h e r e a f t e r falling rapidly ( 16) . Urinary folate as measured by microbiological a s s a y e x c r e t ion a f t e r o r a l administration r e a c h e s a maximum in 2 - 3 hours and r e t u r n s to background values a f t e r 6 hours ( 27) . labelled folic acid (III) is

When tritium

a d m i n i s t e r e d to human s u b j e c t s the m a j o r

portion is e x c r e t e d in the f i r s t 24 hours (33).

Thereafter small

amounts of radioactivity a r e e x c r e t e d each day up to 7 days (33).

Thus

f r o m m e a s u r e m e n t s of serum and urinary folate l e v e l s in man it appears that folate exist in two distinct metabolic pools.

A pharmacokinetic

analysis of serum and urinary folate after o r a l folic acid (III) a d m i n i s tration showed l o s s of serum folate and urinary folate e x c r e t i o n to have f i r s t order kinetics (Fig. 1) (16,27). life of

- 2 hours.

The serum folate l o s s had a half-

No evidence could be obtained from the p h a r m a c o -

388

TIME

(mins)

FIGURE

1

389

COOH

CHO

X / N

NH-

if

H2N

CONH

CH I

C H ,¿ I

C H ,2 I COOH

EZEL

CH — ^ Y

CONH

COOH ÇH I C H ,2 I CHZ ¿OOH

"XVTÏÏ

R -

COOH NH C H i

R = a folate residue

C H ,£ i CH, i

CO

COOH —

NH C H N CH, I C CH2

YTY

COOH

COOH i

H

C O NH CH i

CH2 I C H .¿ i

CO

COOH —

NH C H N

C H 2£ I

CH2 XX

COOH

390 kinetic analysis of a storage pool in rapid exchange with the serum folate pool.

In man t h e r e is a r e n a l threshold of c a . 10 n g / m l . for

folate (34). The existence of two metabolic pools with such different kinetic c h a r a c t e r i s t i c s allows the study of the metabolic pathways in each pool.

In

studies on the rat the metabolites appearing in the f i r s t 24 h r . urine after o r a l administration r e p r e s e n t the metabolism of the f i r s t pool. Urinary metabolites in the second, third and l a t e r days r e p r e s e n t second pool m e t a b o l i s m .

L i v e r metabolites three days and s i x days after o r a l

administration again r e p r e s e n t second pool m e t a b o l i s m . 14 In the rat 2 -

C - f o l i c acid (III) gives f i r s t 24 h r . urinary radioactive

folate containing (a) folic acid (III), (b) 5-methyltetrahydrofolic acid (I), (c) 10-formyltetrahydrofolic acid (II) and (d) 4 a - h y d r o x y - 5 - m e t h y l t e t r a hydrofolic acid (XVI) (31, 35).

T h i s last product is not an analytical

14 artifact.

2-

C - 1 0 - F o r m y l t e t r a h y d r o f o l i c acid (II) gives 5 - m e t h y l t e t r a -

hydrofolic acid (I) as the principal f i r s t 24 h r . urinary metabolite (13). 14 2 - C-5-Methyltetrahydrofolic acid (I) gives 5-methyltetrahydrofolic acid (I) (14). 5- 14 C-5-Methyltetrahydrofolic acid (I) gives radioactive metabolites but no radioactive 5-methyltetrahydrofolic acid (I) (13). 2 14 C - 5 - F o r m y l t e t r a h y d r o f o l i c acid (XVII)gives only s m a l l amounts of 5 methyltetrahydrofolic acid (I), the other products being derived from 14 e x c r e t e d unchanged 5-formyltetrahydrofolic acid(XVII)(13). 2- C-10formylfolic acid (X) at low dose gives only breakdown products and no folates (13). In studies on man the metabolites appearing in the s e r u m and urine define the metabolism of the f i r s t pool.

The r e s u l t s obtained a r e s i m i l -

a r to those in the rat except that 10-formylfolic acid (X) is not broken down to s i m p l e r products but r e m a i n s unmetabolised (16) and 5 - f o r m y l tetrahydrofolic acid(XVII)is efficiently converted to 5 - m e t h y l t e t r a h y d r o folic acid (I) (16).

T h i s l a t t e r effect is due to rapid cyclisation in the

391 acid human stomach of 5-formyltetrahydrofolic acid(XVII)to

10-methyl

-idine-tetrahydrofolic ac id (X VIII) which then f o r m s 1 0 - f o r m y l t e t r a h y d r o folic acid (II) in the jejunum (36).

The r a t stomach is not sufficiently

acid to promote c y c l i s a t i o n of 5-formyltetrahydrofolic acicfXVII). studies on the s e r u m folate pool in man show that it c o n s i s t s of two folates;

a

m i n o r component, 10-formyltetrahydrofolic acid (II) and a m a j o r compon -ent, 5-methyltetrahydrofolic acid (I) (16).

Both folates a r e in dynamic

metabolic equilibrium and in health the level of 10-formyltetrahydrofolic acid (II) is kept constant (O. 8 n g / m l ) while the level of 5 - m e t h y l t e t r a hydrofolic acid (I) r i s e s and falls a s it a c t s as a s t o r a g e f o r m ( 1 6 ) . Studies in man and the r a t show that the s e r u m levels of folates have a diurnal variation (2 7, 37). While it has not yet been possible to determine second pool metabolism in m a n detailed studies have been c a r r i e d out in the r a t .

4a-Hydroxy -

5 - m e t h y l - 4 , 5 , 6 , 7-tetrahydrofolic acid (XVI) a metabolite present in the f i r s t 24 hour urine after o r a l folic acid b e c o m e s the m a j o r m e t a b o l ite in the second and third day urines (Table 5/ (31, 35).

This compound

is t h e r e f o r e the m a j o r metabolite of the relainal folate pool.

The failure

of other w o r k e r s to identify the p r e s e n c e of this metabolite (38) is due to t h e i r use of ion exchange chromatography on DE52 cellulose, a system which does not easily s e p a r a t e 10-formyltetrahydrofolic acid (II) f r o m 4 a - h y d r o x y - 5 - m e t h y l - 4 , 5 , 6 , 7-tetrahydrofolic acid (XVI).

In our stud-

ies effective separation was achieved by Sephadex G15 and thin l a y e r chromatography (31, 35).

Rat l i v e r r e t a i n s substantial amounts of

radioactivity t h r e e and six days a f t e r o r a l administration (Table 4 )(13, 31, 32) and this allows a study of folate metabolism within the second 14 pool. A f t e r o r a l 5 - C - 5 - m e t h y l t e t r a h y d r o f o l i c acid (I) ¡Liver folate a f t e r t h r e e days is 5-methyltetrahydrofolic acid (I) (13). L i v e r folate 14 t h r e e days after 2 - C - 1 0 - f o r m y l t e t r a h y d r o f o l i c acid (II) is 5 - m e t h y l 14 tetrahydrofolic acid (I) (13). O r a l 2 - C - 5 - f o r m y l t e t r a h y d r o f o l i c acid (XVII) gives a s m a l l proportion of total radioactivity a s 5 - m e t h y l t e t r a -

592 5

TABLE

Distribution of Urinary Metabolites after Oral Folic Acid'a' (III)

4a-hydroxy-5-methyltetrahydrofolic acid (XVI)

DAY

HN

4-methyl tetrahydrofolic acid(l)

(b)

1

43

2

83

14

3

67

9

29

(a)

oral dose

(b)

as per centage of total radioactivity in urine.

0

?H3 Y

T

h

A X HjN^N'^hT

32 yg/kg:

Ov H N

mean of four animals

?H3

?H3

O

X / N ^ R flf Tt

" " HXN s k r J H J N ^ N ^^ ^ N J^ j

addition _

HN

^f

ofaddition proton to H N L J form stable HjN^N^^N epimer

(xnz)

COOH R = -CH,NH { % CO N H C H (CHj) C O O H

FIGURE

(b)

2

(IEZ)

R H

393 hydrofolic acid (I) in l i v e r t h r e e days l a t e r i n c r e a s i n g to about 50% of 14 the t o t a l r a d i o a c t i v i t y 6 days l a t e r . (13 V 2 - C - 1 0 - F o r m y l f o l i c acid (X) a d m i n i s t e r e d o r a l l y gives r a d i o a c t i v e 10-formylfolic acid (X) and folic acid (III) in the l i v e r t h r e e days l a t e r changing to folic acid (III) and 5 - m e t h y l t e t r a h y d r o f o l i c acid (I) at six days (13).

T h u s in the r a t the

second pool of r e t a i n e d f o l a t e s d i f f e r s f r o m the f i r s t pool not only in kinetic behaviour but also in the capacity to m e t a b o l i s e slowly 1 0 - f o r m y l - f o l i c acid (X) and 5 - f o r m y l t e t r a h y d r o f o l i c acid(XVII)to 5 - m e t h y l t e t r a hydrofolic acid (I); a p r o p e r t y absent f r o m the f i r s t pool. The biological r o l e of the second pool of r e t a i n e d f o l a t e s h a s a t t r a c t e d much interest.

It has been thought that in the n o r m a l a n i m a l it a c t s a s

a s t o r a g e f o r m f o r folate m o n o g l u t a m a t e s .

However when the m e t a b o l -

ite, 4 a - h y d r o x y - 5 - m e t h y l - 4 , 5, 6, 7 - t e t r a h y d r o f o l i c acid (XVI), d e r i v e d f r o m the s t o r a g e f o r m is o r a l l y a d m i n i s t e r e d to r a t s it is a b s o r b e d and e x c r e t e d unchanged (14).

O t h e r w o r k e r s have suggested that the r e t a i n -

ed f o r m s a r e folate " p o l y g l u t a m a t e s " (XIX) and that t h e s e a r e the effect -ive coenzymes.

W h e r e the r a t e of f o r m a t i o n of " p o l y g l u t a m a t e s " h a s

b e e n studied a s in the monkey (39) it h a s b e e n shown to be slow, r e q u i r ing between 1 - 2 days f o r completion.

The r a t e of c o n v e r s i o n of 10-

f o r m y l t e t r a h y d r o f o l i c acid (II) to 5 - m e t h y l t e t r a h y d r o f o l i c acid (I) and the exchange of the methyl group of the l a t t e r a r e v e r y r a p i d in the r a t and m a n (13,16,17) Thus on kinetic grounds the m a j o r enzymic i n t e r c o n v e r s i o n s m u s t p r o c e e d without the p r i o r f o r m a t i o n of " p o l y g l u t a m a t e s " . The monoglutamate f o r m s t h e r e f o r e a p p e a r to function a s c o e n z y m e s . 5 - M e t h y l t e t r a h y d r o f o l i c acid (I) r e a d i l y l o s e s e l e c t r o n s to c u p r i c ions, f e r r i c ions, quinones and oxygen to f o r m 5 - m e t h y l - 5 , 6 - d i h y d r o f o l i c acid (XIV) (25, 26) and t h i s is r a p i d l y r e d u c e d to 5 - m e t h y l t e t r a h y d r f o l i c acid (I) by r e d u c i n g agents such a s a s c o r b i c acid.

5 - M e t h y l - 5 , 6 -dihydro

-folic acid (XIV) is unique a s the only stable oxidation p r o d u c t of f o l a t e s which is r e a d i l y r e d u c e d to the o r i g i n a l t e t r a h y d r o compound.

Thus 5 -

m e t h y l t e t r a h y d r o f o l i c acid in the r e t a i n e d f o l a t e s could p a r t i c i p a t e in

394 electron transport reactions.

This view is supported by the occurrence

of 5-methyl-5, 6 -dihydrofolic acid (XIV) in the serum of pernicious anaemia patients and by the formation of 4a-hydroxy-5-methyltetrahydrofolic acid (XVI) as the excreted metabolite of the retained folates.

4a-

Hydroxy-5-methyltetrahydrofolic acid (XVI) could be formed by the addition of water to 5-methyl-5, 6-dihydrofolic acid (XIV). When the 50 : 50 14 diastereoisomeric mixture of 5- C-5-methyltetrahydrofolic acid (I) is orally administered to the rat no radioactive 5-methyltetrahydrofolic acid (I) appears in the urine (13).

Since the transport of 5-methyltetra-

hydrofolic acid (I) a c r o s s the gut is a passive transport p r o c e s s (9) it is unlikely that the biologically inactive diastereoisomer is excluded. Both d i a s t e r e r o i s o m e r s of 5-methyltetrahydrofolic acid (I) must t h e r e fore enter the folate cycle.

This probably occurs by oxidation to 5-

methyl-5, 6-dihydrofolic acid (XIV), epimerisation about C6 to form the biologically active form and reduction to 5-methyltetrahydrofolic acid (I) (Figure 2 ).

These experimental observations again support the view

that 5-methyltetrahydrofolic acid (I) can participate in electron transport p r o c e s s e s , by oxidation to 5-methyl-5, 6-dihydrofolic acid (XIV). When the folates present in animal t i s s u e s are assayed microbiologically it is found that only a small proportion of the folate is immediately available to L. caaei.

The major portion of the folate only b e c o m e s

available to L. c a s e i after digestion with the enzyme conjugase.

As

this behaviour is observed with synthetic polyglutamates (XIX) it is often concluded that the conjugase r e l e a s e d L. c a s e i active compounds are polyglutamates.

Other explanations however are possible.

These

compounds could be folates strongly bound to as some yet unidentified species;

s p e c i e s which do not r e l e a s e their bound folate to L. c a s e i . To

elucidate the polyglutamate problem further and to avoid the ambiguities of microbiological a s s a y a number of workers have assayed the radioactive folates present in monkey and rat liver 24 and 48 hours after oral folic acid (III ) (39,41).

In this approach large radioactive amounts of

395 tritiated

f o l i c a c i d (9, 3 ' 5 - H) a r e o r a l l y a d m i n i s t e r e d , t h e l i v e r e x c i s e d

i m m e d i a t e l y a f t e r d e a t h and p r o m p t l y d r o p p e d into b o i l i n g a s c o r b i c a c i d s o l u t i o n s to d e a c t i v a t e e n d o g e n o u s c o n j u g a s e .

A n a l y s i s of t h e s o l u t i o n s

s o o b t a i n e d show t h e m a j o r p o r t i o n of t h e r a d i o a c t i v i t y t o b e e l u t e d in o r j u s t a f t e r t h e void v o l u m e of Sephadex G15 o r t o a p p e a r at high t u b e n u m b e r on ion e x c h a n g e c h r o m a t o g r a p h y with DE 52.

These radioactive

s p e c i e s do not m a t c h t h e c h r o m a t o g r a p h i c b e h a v i o u r of t h e c o m m o n known f o l a t e m o n o g l u t a m a t e s and t h e y a r e c l a i m e d t o be p o l y g l u t a m a t e f o r m s (39,41).

A v a r i a t i o n of t h i s m e t h o d is t o o x i d i s e t h e r a d i o a c t i v e

c o m p o u n d s i s o l a t e d and i d e n t i f y the r a d i o a c t i v e f r a g m e n t s a s p - a m i n o b e n z o y l p o l y g l u t a m a t e s (XX )

(39).

Using these methods various

g r o u p s h a v e c l a i m e d t o show t h e p r e s e n c e of f o l a t e p o l y g l u t a m a t e s (XIX) in r a t l i v e r (41) and b a c t e r i a (42).

However polyglutamate formation

in m o n k e y l i v e r p r o c e e d s b y s t e p w i s e a d d i t i o n of g l u t a m i c a c i d u n i t s a n d i s s l o w r e q u i r i n g about t w o d a y s f o r c o m p l e t i o n of p o l y g l u t a m a t e s y n t h e s i s (39).

In m o n k e y k i d n e y although f o l i c a c i d i s r e t a i n e d in t h e k i d -

n e y no p o l y g l u t a m a t e s c a n b e d e t e c t e d u n t i l a f t e r t h e f i r s t 24 h o u r p e r i o d (39).

In b a c t e r i a t h e a c c u m u l a t i o n of f o l i c a c i d a g a i n s t a c o n c e n t r a t i o n

g r a d i e n t d o e s not r e q u i r e p o l y g l u t a m a t e f o r m a t i o n a l t h o u g h s o m e p o l y g l u t a m a t e i s f o r m e d (42). A l t h o u g h t r i t i a t e d f o l i c a c i d (III) is thought to f o r m p o l y g l u t a m a t e s on 14 i n c u b a t i o n with l y m p h o c y t e s 5 - C - 5 - m e t h y l t e t r a h y d r o f o l i c a c i d (I) a p p a r e n t l y d o e s not (43).

Although other i n t e r p r e t a t i o n s a r e p o s s i b l e

t h e a u t h o r s c o n s i d e r e d t h a t t h e s e r e s u l t s show t h a t p o l y g l u t a m a t e f o r m a t i o n p r o c e e d s f r o m s o m e m e t a b o l i t e in t h e f o l a t e c y c l e o t h e r t h a n 5 m e t h y l t e t r a h y d r o f o l i c a c i d (I).

If t h i s is s o t h e » t h e d e m e t h y l a t i o n of

5 - m e t h y l t e t r a h y d r o f o l i c a c i d 3(I) is a 14 s t e p not r e q u i r i n g p r i o r p o l y g l u t a m -ate formation. T h e u s e of H and C - l a b e l l e d f o l i c a c i d (I) t o r e v e a l t h e p r e s e n c e of p o l y g l u t a m a t e s (XXI) in r a t l i v e r w a s f i r s t u s e d at t h e U n i v e r s i t y of A s t o n in 1970. (14); s o m e t i m e in a d v a n c e of t h e p u b l i c a t i o n s of o t h e r s (39, 41).

H o w e v e r , b e c a u s e of d i s c r e p a n c i e s b e t w e e n

396 the c h r o m a t o g r a m s obtained by using the ^ C and ^H l a b e l l e d folic acid 14 (III) t h i s work was not published. When 2 - C - 1 0 - f o r m y l f o l i c a c i d (X) was o r a l l y a d m i n i s t e r e d to r a t s and the l i v e r s a s s a y e d a f t e r 3 days f o r p o l y g l u t a m a t e s by the a c c e p t e d technique the DE 52 ion exchange c h r o m atography gave only two compounds, subsequently identified by thin l a y e r chromatography a s 1 0 - f o r m y l f o l i c acid (X) and f o l i c acid (III) and no polyglutamates (44).

Thus substantial amounts of folate can be r e t a i n e d

by the l i v e r

without (a) polyglutamate f o r m a t i o n or (bl)14r e d u c t ion of the pteridine ring. Again after the a d m i n i s t r a t i o n of 5 - C - 5 -

m e t h y l t e t r a h y d r o f o l i c acid the three day l i v e r on a n a l y s i s f o r p o l y g l u t a m a t e s gave only one compound on DE52 ion exchange c h r o m a t o g r a p h y . T h i s was identified a s 5 - m e t h y l t e t r a h y d r o f o l i c a c i d (I) by thin layer 14 chromatography (44). P o l y g l u t a m a t e s w e r e absent. 2 - C-folic acid (III) given o r a l l y to the rat gave DE52 c h r o m a t o g r a m s in which p e a k s c h a r a c t e r i s t i c of p o l y g l u t a m a t e s w e r e absent and 5 - m e t h y l t e t r a h y d r o f o l i c acid (I) was the m a j o r component (Figure 3) (44).

A similar exper

- i m e n t in which 9, 3 ' 5 - H - f o l i c acid 14 (III) was u s e d gave DE52 c h r o m a t o g r a m s different f r o m t h o s e with 2 - C - f o l i c a c i d (III) and having a m a j o r peak of r a d i o a c t i v i t y in the r e g i o n a s s i g n e d to p o l y g l u t a m a t e s by o t h e r s . 3 (Figure 3 ) (44). Thus the s p e c i f i c u s e of H - f o l i c a c i d (III) a p p e a r s to be the only method by which the p r e s e n c e of p o l y g l u t a m a t e s can be 14 detected. No e v i d e n c e f o r p o l y g l u t a m a t e s c a n be obtained u s i n g C14 f o l i c acid (III) or other C - l a b e l l e d f o l a t e s on DE52 c o l u m n s . These r e s u l t s t h e r e f o r e s u g g e s t e d that 9, 3 ' 5 - f o l i c acid (III) exchanged t r i t i u m in the b i o l o g i c a l s y s t e m and that the e v i d e n c e f o r p o l y g l u t a m a t e s derives f r o m an analytical a r t i f a c t . A f t e r o r a l a d m i n i s t r a t i o n of doubly l a b e l l e d 2 - 14 C-9,3'5 1 - 3 H - f o l i c a c i d (III) to the rat the f i r s t s i x hour urine gave a g r e a t e r p e r c e n t a g e r e c o v e r i e s of ^H than ^ C , the 6 - 2 4 hour 14 3 urine gave a much l a r g e r p e r c e n t a g e r e c o v e r y of H than C and s i m i l a r r e s u l t s w e r e obtained with the 24 - 48 hour u r i n e . (35, 37). Ion exchange chromatography on DE 52 c e l l u l o s e and g e l p e r m e a t i o n

397

Tube number

^H levels for fractions from a DEAE-cellulose chromatogram of rat liver extract, from a rat given PteGlu-(3',5',9- 3 H).

Tube number 14 C levels for fractions from a DEAE-cellulose chromatogram of rat liver extract, from a rat given PteGlu-(2- 14 C).

FIGURE

3

398 c h r o m a t o g r a p h y on Sephadex G15 of both u r i n e and l i v e r ( a f t e r hot e x t r a c t 3 14 - i o n ) gave d i f f e r e n t c h r o m a t o g r a p h i c p a t t e r n s of H and C (37). 14 On Sephadex G15 c h r o m a t o g r a m s the

C l a b e l l e d compounds

a p p e a r e d in the void v o l u m e o r s h o r t l y a f t e r .

However a f t e r r e c o v e r y

f r o m G15 c o l u m n s such p e a k s b e h a v e d a s m o n o g l u t a m a t e s on D E 5 2 . T h i s b e h a v i o u r on Sephadex G15 i s p r o b a b l y due to c o m p l e x f o r m a t i o n . T h e s e 3 3 r e s u l t s c l e a r l y show t h a t H - f o l i c a c i d (III) e x c h a n g e s H with the b i o l 3 ogical system.

T h e u s e of

H - f o l i c a c i d (III) t o m e a s u r e f o l a t e m e t a b o l -

i s m and pools i s t h e r e f o r e u n r e l i a b l e . Since "polyglutamate" formation 3 14 c a n only be d e m o n s t r a t e d with H - f o l i c a c i d (III) and not C-labelled 14 folic acid or C -labelled folates they a r e analytical artifacts derived 3 from H exchange. F o l i c a c i d (III) r e a c t s with C F C O O D / D 0 m i x t u r e s at 4 0 ° C to give 3», 5L 2 H-folic acid (Figure 4 ) (45). T h u s t h e r e i s a good c h e m i c a l m o d e l f o r 3 3 H e x c h a n3 g e . In addition the 9, 3 ' 5 ' - H - f o l i c a c i d (III) u s e d could e x c h ange t h e H isotope at p o s i t i o n 9. E a r l i e r w o r k e r s (46) have c l a i m e d that the d i s t r i b u t i o n of individual f o l a t e s within t i s s u e s v a r i e d with the r a t e of c e l l d i v i s i o n .

Their results

showed that in t i s s u e s with slow r a t e s of c e l l division ( e . g . n o r m a l l i v e r ) the m a j o r f o l a t e was 5 - m e t h y l t e t r a h y d r o f o l i c a c i d (I).

In t i s s u e s with

i n c r e a s e d r a t e s of c e l l division ( i n t e s t i n e , W a l k e r 256 c a r c i n o m a , M u r p h y - S t u r m l y m p h o m a ) t h e proportion of f o l a t e a s 5 - m e t h y l t e t r a h y d r o f o l i c a c i d (I) d e c r e a s e s until it b e c o m e s a m i n o r component in the t i s s u e with t h e highest 1 r a t e of c e l l d i v i s i o n .

14

of u r i n a r y f o l a t e s a f t e r o r a l a d m i n i s t r a t i o n of 2 -

A p r e l i m i n a r y analysis C - f o l i c a c i d to r a t s

with l a r g e implanted W a l k e r 256 c a r c i n o m a s showed a different d i s t r i b ution f r o m u r i n a r y f o l a t e s in the n o r m a l r a t . t e t r a h y d r o f o l i c a c i d (XVI) was m u c h r e d u c e d (

4 a - H y d r o x y - 5 - m ethyl 44

).

F u r t h e r work

h a s shown that a f t e r o r a l f o l i c a c i d in n o r m a l r a t s t h e m a j o r 24 h o u r u r i n a r y f o l a t e i s 5 - m e t h y l t e t r a h y d r o f o l i c a c i d (I) while in r a t s with l a r g e

399

COOH C H 2 NH $

V C O N H CH CH& CHZ COOH

CH 2 NH

FIGURE 4

COOH I CONH CH I CH» I CHO I COOH

CF 3 COOD/D 2 O at 4 0 ° C .

400 implanted Walker 256 tumours the major 24 hour urinary folate is 10formyltetrahydrofolic acid (II).

Analysis of liver and tumour folates in 14 r a t s with Walker 256 tumours after oral 2 - C-folic acid with DE 52 ion exchange chromatography showed the major folate to be 1 0 - f o r m y l t e t r a -

hydrofolic acid (II) (35, 3 7)In normal rat liver the major folate is 5 methyltetrahydrofol'ic acid (I).

Thus the e a r l i e r claims (46) have been

confirmed in the r a t . Similar detailed studies in man are not yet possible.

However it has

been shown in man that the serum 5-methyltetrahydrofolic acid (I) and 10-formyltetrahydrofolic acid (II) a r e in rapid dynamic metabolic equilibrium (16) and that the serum level of 10-formyltetrahydrofolic a c i d ( n ) is kept at a constant value of 0. 8 ng/ml in health. -spread lymphoma gave a value of 4. 0 ng/ml;

One subject with wide

41 subjects with leukaemia

gave a significantly increased serum value of 1.23 i 0. 07 n g / m l . for 10formyltetrahydrofolic acid (II) despite total serum folate values l e s s than normal (Table 6 ) (16).

Thus as in the rat malignant disease is

accompanied by an increase in the levels of 10-formyltetrahydrofolic acid.

Similar i n c r e a s e s in the serum levels of 10-formyltetrahydro-

folic acid (II) despite the lowered level of total serum folate a r e found in adult coeliac disease, rheumatoid arthritis and regional enteritis (Table 6) (16, 48);

all disease states in which there is an enhanced rate of cell

division. In scurvy the serum level of ascorbate falls to z e r o .

Thus the serum

folates of scorbutics will be more rapidly oxidised than in the normal subject.

Studies at low concentrations of ascorbate show that ascorbate

r e t a r d s oxidation by a factor of about two (21).

10-Formyltetrahydro-

folic acid (II) will be oxidised c a . ten times faster than 5 - m e t h y l t e t r a hydrofolic acid (I) and will be irreversibly oxidised to the non utilisable 10-formylfolic acid (X) (Fig.

5

).

Thus in scorbutics there will be a

steady depletion of the folate pool resulting in anaemia.

Studies in a

group of scorbutics has shown that the principal urinary folate is 10-

401

TABLE

6

MEAN SERUM VALUES OF FOLATES IN HUMAN SUBJECTS

No of subjects

Subjects

10-formyl 5 methyltetrahydrofolic tetrahydrocn/i \ ( ) (c) . ,,TT. (a) (b) (c) folic acid (1) acid(II)

Normal

51

4.80+ 0.28

0.78 + 0.03

Leukaemia

41

3.70+ 0.37

1.23 + 0.07

Adult coeliac disease

56

4.59+ 0.46

1.1 +

Rheumatoid Arthritis

39

2.13+ 0.29

1.67 + 0.2

(a)

as determined from total S. faecalis value

(b)

mean value _+ S.E.M.

(c)

value as ng/ml

5MeTHF (1)

7

10 CHOTHF

irreversible — oxidation

(11)

10 CHOFA (X)

Figure 5

0.06

402

f o r m y l f o l i c acid (X) in contrast to normal subjects where the m a j o r urinary folate is 5-methyltetrahydrofolic acid (I) (49).

Administration

of ascorbic acid to a scorbutic subject changed the dominant urinary folate f r o m 10-formylfolic acid (X) to 5-methyltetrahydrofolic acid (I) (49). The author wishes to express his thanks to the following people f o r their assistance and co-operation: D r . P . A . B a r f o r d , D r . J. R . G. Beavon, Dr." W . T . C o o k e , D r . E . Dransfield, D r . I. T . Johnson, D r . M . L u c a s , P r o f e s s o r A . J. Matty, D r . V . Melikian, M r . R . J. L e e m i n g , M i s s H. Portman Graham, D r . A . J. P e a r s o n , M r . A . J . R o b b , D r . I. H. Rosenberg, D r . K . J. Saunders, Dr.~M. E.Smith, D r . R. Stocks-Wilson, M r . F . J.Staff, M r . A . Razzaque and M r . K . Ratanashien, and to the following organisations f o r financial support: The Cancer Research Campaign, The Medical Research Council, The Royal Society, The Science Research Council, The University of Aston in Birmingham and The W e l l c o m e T r u s t .

REFERENCES 1.

Santini, R. , B r e w s t e r , C. and Butterworth, C. E. Jr. : The D i s tribution of F o l i c A c i d A c t i v e Compounds in Individual Foods. A m . J.Clin. Nutr. , 14, 205 - 220 (1964).

2.

F o r d , J E . , Knaggs, G. S. , Salter, D N. and Scott, K. J. ^Folate Nutrition in the Kid. B r i t . J. N u t r . , 2 7 5 7 1 - 583 (1972).

3.

P e r r y , J. : Folate Analogues in N o r m a l Mixed Diets. B r i t . J . H a e m a t o l . , _21i_435 - 441 (1971).

4.

Rosenberg, I . H . , Solomons, N . , Wagonfeld, J . , Russell, R. and Godwin, H. : (This Symposium).

5.

B l a i r , J. A . and Matty, A . J. : A c i d M i c r o c l i m a t e in Intestinal A b sorption. Clinics in Gastoenterology, 3, 1, 183 - 197.

6.

B l a i r , J. A . , Ratanashien, K . and L e e m i n g , R . J. : Intestinal A b sorption of Tetrahydrobiopterin and P t e r o i c A c i d in Man. Gut. 15, 832, (1974).

7.

B l a i r , J. A . and L e e m i n g , R . J . Unpublished Observations (1975).

403 8. Lucas, M . L . , Schneider, W . , Haberich, F . J . and Blair, J. A. : Direct Measurement by pH-Microelectrode of the pH Microclimate in Rat Proximal Jejunum. Proc. Roy. Soc. (B) (in p r e s s ) , (1975). 9. Blair, J. A . , Razzaque, A. and Matty, A. J. : Uptake of 5-Methyltetra -hydrofolic Acid in Rat Jejunum. J. Physiol. (Lond.) (in p r e s s ) (1975). 10. Blair, J. A . , Johnson, I . T . and Matty, A . J . , Uptake of Folic Acid by Various Preparations of Isolated Rat Jejunum Cells (in preparation) (1975). 11. Benn, A . , Swan, C. J. J . , Cooke, W . T . , Blair, J . A . , Matty, A . J . and Smithj M. E. Effect of Intraluminal pH on Absorption of Folic Acid. Brit. Med. J. i, 148 - 150, (1971). 12. Blair, J. A . , Ratanashien, K. and Leeming, R. J . : Unpublished Observations (1975). 13. Beavon, J . R . G . and Blair, J. A. : The Metabolism of Some Folates i n t h e R a t . B r i t . J . N u t r . (in p r e s s ) (1975). 14. Dransfield, E . , P h . D . T h e s i s , University of Aston in Birmingham. The Fate of Folic Acid and Related Compounds i n the Rat (19 72). 15. Blair, J. A . , Lucas, M . L . and Matty, A. J . : Acidification in the Rat Proximal Jejunum. J. Physiol. (Lond. ) 245, 333 - 350, (1975). 16. Ratanashien, K. , Blair, J . A . , Leeming, R. J . , Cooke, W . T . and Melikian, V. : Folates in Human Serum. J. c l i n . P a t h . , _27, 875 879, (1974). 17. Nixon, P . F . and Bertino, J. R. : Impaired Utilisation of Serum Folate in Pernicious Anaemia. J. c l i n . Invest., 51, 1431 - 1439, (1972). 18.Blair, J . A . and Pearson, A. J. : Kinetics and Mechanism of the Autoxidation of the 2-Amino-4-hydroxy-5, 6, 7, 8-tetrahydropteridines. J. Chem.Soc. Perk. 2 . , 80 - 88 (1974). 19. Pearson, A. J. : Kinetics and Mechanism of the Autoxidation of T e t r a hydropterins. Chem. and Ind., 233 - 239, (1974). 5 10 20.Robinson, D.R. : The Nonenzymatic Hydrolysis of N , N - Methenyl -tetrahydrofolic Acid and Related Reactions. Methods in Enzymology XVIIIB, 716 - 72 5, (1971). 21.Blair, J. A. and Pearson, A. J."': Unpublished data (1974). 22.Butterworth, C . E . , Santini, R. and F r o m m e y , W. B. : The P t e r o y l glutamate Components of American Diets as Determined by Chromatographic Fractionation. J. c lin. Invest. 1929 - 1939,(1963). 23.Lewis, D . T . : Some Current Problems in Food Analysis, Chemistry, Medicine and Nutrition Symposium, 75 - 77. The Royal Institute of Chemistry, London. (1966).

4-04 24.

Bertino, J . R . , Perkins, J. P. and Johns, D. G. : Purification and Properties of Dihydrofolate Reductase from Ehrlich Ascites Carcinoma Cells. Biochemistry, 4, 839 - 846 (1965).

25.

Blair, J. A . , Pearson, A . J . and Robb, A . J . : Autoxidation of 5Methyltetrahydrofolic Acid. J.Chem.Soc. Perk. 2, 18 - 21,(1975).

26.

Blair, J. A . and Robb, A . J . : Unpublished observations (1974).

27.

Blair, J . A . , Cooke, W . T . , Leeming, R.J. and Ratanasthien, Jli Unpublished observations, (1975).

28.

Herbert, V. and Zaluksy, R. : Inter-relations of Vitamin B12 and Folic Acid Metabolism. J. clin. Invest., 41, 1263 - 1275, (1962).

29.

Chanarin, I. and P e r r y , J. : Metabolism of 5-Methyltetrahydrofolate in Pernicious Anaemia. Brit. J. Haematol., _14, 297 - 301. (196 8) .

30.

Thenen, S . W . , Shin, Y . S. and Stokstad, E. L . R. : The Turnover of Rat Liver Folate Pools. P r o c . Soc. Exp. Biol. Med. 142, 638 641. (1973).

Si.

Blair, J. A . and Dransfield, E. : The Urinary Excretion of Orally Administered Pteroyl-L-glut amic acid by the Rat. Biochem. J., 123, 907 - 914, (1971).

32.

Blair, J. A. and Staff, F . J . : Unpublished observations (1975).

33.

Landon, M . J . , Eyre, D.H. and Hytten, F . E . , Transfer of Folate to the Foetus. Brit. J. Obstetrics and Gynaecology, 82, 12 - 19. (1975). ~~

34.

Johns, D.G. , Sperti, S. and Burgen, A . S. V. : The Metabolism of Tritiated Folic Acid in Man. J. clin. Invest., 40, 1684 - 1695.

35.

Barford, P . A . and Blair, J. A . (This Symposium).

36.

Beavon, J . R . G . and Blair, J. A . : The pH- dependent rearrangements of formyltetrahydrofolates and their nutritional implications. Brit. J. Nutr. , _28, 385 - 390 (1972).

37.

Barford, P. A . and Blair, J. A. : unpublished observations (1975).

38.

Vidal, A . and Stokstad, E. L R. : Urinary Excretion of 5-Methyl tetrahydrofolate and Liver S-Adenosylmethionine Levels of Rats Fed a Vitamin Bl2 - Deficient Diet. Biochim. biophys. Acta, 362, 245 - 257 (1974).

39.

Brown, J. P . , Davidson, G . E . and Scott, J. M. : The Identification of the Forms of Folate Found in the L i v e r , Kidney and Intestine of the Monkey and their Biosynthesis from Exogenous Pteroyl -glutamate (Folic Acid). Biochim. biophys. Acta., 343, 78 - 88, (1974).

405 40.

Blair, J. A . , L e e m i n g , R. J . , Ratanashien, K. and Shadforth, M . : Unpublished Observations (1975).

41.

Shin, Y . S . and Stokstad, E . L . R . Identification of F o l i c Acid Compounds in Rat L i v e r . B i o c h e m . Biophys. R e s . C o m m u n . , £7, 35 - 43 (1972).

42.

Buehring, K. U . , Tamura, T . and Stokstad, E . L. R . : F o l a t e G o e n z y m e s of Lactobacillus c a s e i and Streptococcus f a e c a l i s . J. biol. C h e m . , 249, 1081 - 1089. (1974).

43.

Lavoie, A . , Tripp, E . and Hoffbrand, A . V . : Methylfolate M e t a b o l i s m in Vitamin B12 . D e f i c i e n c y . Clin. Sci. Mol. M e d . , 47, 617 - 630 (1974).

44.

Beavon, J . R . G . , P h . D . T h e s i s , University of A s t o n in B i r m i n g h a m . Folate and Tetrahydrofolate Metabolism in the Rat (1973)^

45.

Rosenberg, I . H . , Palladino, L . , Hachey, D. and B l a i r , Unpublished Observations (1974).

46.

Sotoyobashi, H . , R o s e n . F . and Nicol, C . A . , Tetrahydrofolate Cof a c t o r s in T i s s u e s Sensitive and R e f r a c t o r y to Amethopterin, B i o c h e m . , _5, 3878 - 3883, (1966).

47.

B l a i r , J. A . , Ratanasthien, K . , L e e m i n g , R . J.,Cooke, W . T . and Melikian, V . : Human Serum F o l a t e s in Health and D i s e a s e . A m e r . J. Clin. N u t r . , _27, 441, (1974).

48.

Stokes, P . L . , Melikian, V . , L e e m i n g , R . J . , Portman-Grahain, H . , B l a i r , J. A . and Cooke, W . T . : F o l a t e Metabolism in Scurvy, A m . J . C l i n . N u t r . , 28, 126 - 129, (1975).

J.A.:

DISCUSSION Wagner: In response to your implication that most studies which have been carried out to study folic acid uptake by isolated systems have been in error because of the long periods of incubation which have been used, I must mention work which we have presented at the 1974 meeting of the FASEB. In these studies we showed that natural 5-methyl-THF was carried into the Porcine Chlorid Plexur by a carrier mediated system. The period of measurement was 5 minutes. This work was confirmed recently by Lorenzo et al. in an article appearing in Science recently. However, I should also point out that preliminary experiments we are now carrying out using the isolated cell preparation of rat liver indicate that both folate and 5-methyl THF enter these cells by simple diffusion. Blair: Our studies relate to intestival transport only. There is now a large literature in this field. The studies using long term incubation periods of 5-methyltetrahydrofolic acid without attention to decomposition were those of Hoffrand and his coworkers.

Red Blood Cell Polyglutamyl Folates in Vitamin B12 Deficiency J. Perry, I. Chanarin and M. Lumb

One of the abnormalities of folate metabolism in vitamin is a decrease in red cell folate concentration (1, 2).

deficiency Red blood cell

folates may be divided into two groups depending on their utilization by Lactobacillus casei during microbiological assay.

One group, those with

from 1 - 3 glutamic acid residues, are immediately available as growth factors for the assay organism, but L. casei cannot utilize the if - 6 glutamyl compounds until these have been converted to short chain forms by the action of gamma glutamyl carboxypeptidase or conjugase.

When

whole blood is prepared for the routine red cell folate assay by dilution in a solution of

ascorbate, plasma conjugase (pH optimum approximately

5) rapidly hydrolyses the long chain forms to monoglutamyl folates, available as growth factors for L. casei.

This assay does not distin-

guish between the relative concentration of long and short chain folates in the red cells.

If however, plasma conjugase is inactivated by dilut-

ing whole blood in boiling ascorbate at pH 7.0, only the 1 - 3 glutamyl folates are measured (3).

The difference between the two assay values

obtained from red cells treated in each of these two ways is a measure of the if - 6 glutamyl folates. This method was used to determine the ratio of 1 - 3 glutamyl to If - 6 glutamyl folates in the red cells of patients with pernicious anaemia, and in normal subjects the 1 - 3 glutamyl folate concentrations were the same in each group, but the if - 6 glutamyl folate concentrations were significantly different, the mean for the vitamin B ^ deficient group being 108 ng/ml, and for controls 203 ng/ml (if). When vitamin B 1 2 is given to patients with pernicious anaemia the red cell folate increases rapidly.

This increase proved to be due to a

rapid restoration of long chain folate concentration, with no change in

408 • — • Pte Glu 4-6 ••—•• Pte Glu 1-3

Days Figure 1.

The effect of vitamin B ^ administration on the red cell polyglutamyl folate concentration in pernicious anaemia.

the 1 - 3 glutamyl folate concentration (Fig.1), thus indicating that vitamin B 1 2 is required directly or indirectly for the synthesis of 1+ - 6 polyglutamyl folates. To determine whether in vitamin B^^ deficiency, there was any qualitative change in folate polyglutamates lysates were chromatographed on an 0.9 x 50 cm. column of DEAE cellulose, using a phosphate buffer gradient elution at pH 6.0.

Five ml. fractions were collected.

Each fraction

eluted from the column was assayed microbiologically and the identity of the peaks of folate activity confirmed by co-chromatography of the lysates with high specific actively tritium labelled methyl polyglutamates of known glutamyl chain length. Bed cell lysates from 6 normal subjects had an almost identical pattern of folate compounds.

The major component was methyl pentaglutamate with

a lesser quantity of tetra- and hexaglutamates, and small peaks of what are presumably mono di and triglutamates (Fig.2).

In B^^ deficiency the

pattern of polyglutamyl folates was similar to that found in normal

¿4-09

5CH3 H4 Pte Glu 5

Fraction number

Figure 2.

Chromatograph of a lysate of normal red cells. The labelled peaks are those whose identity was confirmed by co-chromatography with standard marker compounds.

subjects, methyl pentaglutamate again being the major fraction.

Chroma-

tography of the red cells from a patient with pernicious anaemia after four weeks of B ^ therapy showed increased amounts of the shorter chain polyglutamates, probably representing the folates formed after the first few days of B ^ administration (Fig.3).

In red cells from a severely

folate deficient patient the relative proportions of the different folates were similar to those in normal red cells. There are several views about the metabolic role of long chain folates, but evidence is accumulating that the polyglutamyl form is the active coenzyme.

Most of this work has been done with bacterial systems (5, 6),

but there are two recent reports of work with mammalian cell lines which support our view that the long chain polyglutamyl folates are in fact the coenzyme form of the vitamin.

One of them describes the effectiveness

of dihydropteroylpolyglutamates as substrates for mammalian dihydrofolate reductase (7), and the other, in which a hamster cell line that could not form polyglutamates was shown to have an absolute requirement for adenosine, thymidine and glycine (8), compounds whose synthesis is folate dependent. There are three possible causes for a failure of polyglutamyl folate synthesis in vitamin B._ deficiency in man

-

vitamin B.. could be a

Fraction number

Figure 3«

Chromatograms of red cell lysates from a patient with pernicious anaemia before, and k weeks following, vitamin B 1 2 therapy.

direct participant in polyglutamyl folate synthesis, secondly, indirectly by a failure of transport of folate into cells (9, 10), which may be regulated by conversion of monoglutamyl folate to the polyglutamate coenzyme, and thirdly, by a need to change methyl folate into another folate compound which may be the active substrate for polyglutamate synthesis.

This latter possibility is now being explored by attempting

to determine the physiological folate substrate required for polyglutamyl folate synthesis. Acknowledgements. We wish to thank Dr. K-U. Buehring for the gift of tritium labelled methyl polyglutamates. REFERENCES 1. Cooper, B.A. and Lowenstein. : Relative folate deficiency of erythrocytes in pernicious anaemia and its correction with cyanocobalarain. Blood. 502 (1964).

411

2. Hansen, H.A.: On the diagnosis of folic acid deficiency. Almqvist and Viksell, Stockholm (1964). 3. Shin, G.S., Buehring, K.U. and Stokstad, E.L.R.: Studies of folate compounds in nature. Folate compounds in rat kidney and red blood cells. Arch.Biochem.Biophys. 163 . 211-221+ (1974). 4. Chanarin, I., Perry, J. and Lumb, M.: The biochemical lesion in vitamin B 1 2 deficiency in man. Lancet, 1251-1252 (1974). 5. Curthoys, N.P., and Rabinowitz, J.C.: Formyltetrahydrofolate synthetase. Binding of folate substrates and kinetics of the reverse reaction. J.Biol.Chem. 2i£, 1965-1971 (1972). 6. Kisliuk, R.L., Gaumont, G. and Baugh, C.M.: Polyglutamyl derivatives of folate as substrates and inhibitors of thymidylate synthetase. J.Biol.Chem. 249. 4100-4103 (1974). 7» Coward, J.K., Parameswaran, K.N., Cashmore, A.R., and Bertino, J.B.: 7,8-dihydropteroyl oligo-V-L-glutamates: synthesis and kinetic studies with purified dihydrofolate reductase from mammalian sources. Biochem. I^, 3899-3903 (1974). 8. McBurney, M.U., and Whitmore, G.T.: Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells. Cell. 2, 173-182 (1974). 9. Das, K.C., and Hoffbrand, A.V.: Studies of folate uptake by phytohaemagglutinin-stimulated lymphocytes. Brit.J.Haemat. 19, 459-468 (1970). 10. Tisman, G., and Herbert, V.: B ^ dependence of cell uptake of serum folate: an explanation for high serum folate and cell folate depletion in B 1 2 deficiency. Blood, ±1, 465-469 (1973). DISCUSSION G.M.Brown: Your idea, that B-|2 may be involved in polyglutamate formation, is interesting. I would like to point out, however, that B12 cannot be involved in polyglutamate in E.coli since this baterium neither synthesizes nor needs vitamin B ^ Perry: The function of B 1 2 in bacteria of lower mammals appears quite different from that in the human being - even in primates B-] 2 deficiency does not produce megaloblastosis, for example. Rowe: Comment - Km for 5-methyl-H4 PtGlu^ for B ^ dependent methionine synthetase in rat liver is 1/5 that of 5-methyl ^PtGlu-j. Perry:

This was unknown to me.

Waxman: Could the reticulocyte response to vitamin B-] 2 contribute to the rise m the folyl polyglutamates noted in your studies ? Perry:

This may well be so. We made no effort to separate reticulocytes

412

from the red celll lysates. Niethammer: I would like to make a comment on your second possibility of interaction between vitamin B-j 2 and folate deficiency. You stated that Tisman and Herbert, and Das and Hoffbrand have shown that vitamin B-|? is required for folate transport. But both groups only studied total uptake over a longer period of time and they showed no kinetic data, since metabolism and intracellular binding influence total uptake to a great extent. Their data really do not give a clue for the fact that vitamin B12 plays a role in transport of folates. An activation of the B-j 2~ dependent enzyme methionine synthetase seems to be a better candidate for the interaction between vitamin B-| 2 and 5-methyltetrahydrofolate in pernicious anemia. Perry: If, as seems likely, polyglutamate folate is the active coenzyme form, decrease of 5-methyl THF-transport into cells in vitamin B^ 2 deficiency, as indicated by Das and Hoffbrand and Tisman and Herbert, could be due possibly to failure of polyglutamyl synthesis. Although neither group of workers gave kinetic data, their data in vitamin B-|2 deficient cells were compared with those in normal cells. Our third hypothesis was that methyl THF may not be the folate compound which acts as the precursor of polyglutamyl synthesis. We are aware of the importance of the methionine synthetase reaction, and it is this latter point that we are at the moment investigating.

Novel Urinary Metabolites of Folic Acid in the Rat P.A. Barford and J.A. Blair

INTRODUCTION Blair and Dransfield (1) have reported that, following an 14 oral dose of C folic acid, urinary radioactivity was comr posed of two major metabolites and one minor metabolite. One of the major metabolites was identified as 5-methyltetra hydrofolic acid. ified.

The two other metabolites remained unident-

Further studies (2) identified 10-formyltetrahydro-

folic acid in normal rat urine.

The unidentified metabolite

was readministered to rats and found to be excreted un changed in urine (3).

In a recent paper Vidal and Stokstad

(4) reported the presence of 5-methyltetrahydrofolic acid, lOTformyltetrahydrofolic acid and 5-formyltetrahydrofolic acid in normal rat urine following intraperitoneal inject3 ions of H folic acid. They did not report finding folic acid, or a major unidentified metabolite in rat urine.

The

work reported here is concerned with the identification of an unknown metabolite in rat urine. RESULTS Normal rats, and rats with a Walker 256 carcinoma (both 15014 200g body weight) received oral doses of C folic acid (2 yCi, 78 yg/Kg body weight).

Rats were then placed in

metabolism cages designed for the separate collection of urine and faeces.

Urine, collected into 10 ml. of 0-.05M

phosphate buffer pH 7.0 containing 2% sodium ascorbate and 5mg% dithiothreitol, was collected in 0-6h., 6-24h. and 24-48h. fractions.

Total radioactivity in each sample was

determined and urine samples were then subjected to chromato-

¿m Normal Rats Animal No.

Urine samples 0-6h. 6-24h.

1 2 3 4 5 6 7 8

2.9 4.6 7.7 5.1 4.2 19. 7 1.2 5.3

Average

6.3

24-48h.

Faeces

Liver

Total

4.4 7.2 13.0 4.7 2.4 1.3 0.8 4.4

4.5 7.9 5.9 1.9 1.5 0.9 2.2 2.2

45.4 44.2 52.4 42.8 39.8 24. 1 57. 1 53.0

12. 3 12.5 11.6 20.1 18.7 17.6 17.1 12.5

69.5 66.4 90 . 6 74.6 66.6 63.6 78.4 75.4

4.2

3.4

44.5

15. 3

73.1

Tumour bearing rats 1 2 3 4 5 6 7 8 9 10 11 12 13 rerage

4.2 1.1 6.0 13.4 6.3 12.0 5.1 2.5 7.0 0 8.2 8.2 10.8

15.1 19.9 12.4 4.9 3. 4 6.1 4.3 17.6 20.6 10. 3 9.3 10. 3 13.2

1.5 1.3 1.8 1.1 1.5 2.1 1.8 3.2 0.7 2.1 1.6 2.3 1.8

24. 1 39. 6 33. 5 36. 2 4-7.2 36. 0 52. 8 58. 9 35. 0 34. 7 35. 3 32. 3 26. 1

13.2 18. 4 11.0 10.0 18.8 15.0 13.9 14.1 13.0 16.5 14.0 16.5 18.0

58. 1 80. 3 64. 7 65. 6 77. 2 71. 2 77. 9 92. 3 76. 3 63. 6 68. 4 69. 5 69. 9

6.2

11.2

1. 7

37. 7

14. 7

71. 7

Figure I Distribution of radioactivity

(expressed as percentage of

dose given to animal) in urine, faeces and liver of normal 14 and tumour bearing rats following an oral dose of C folic acid.

415

NaCl) 0.8 M

1.0 (NaCl)

0.8 M

58

Figure

66 74 Sample no (5 mis)

82

86

(2)

D.E.A.E. cellulose chromatograms of urinary folates of normal rats, A, 0-6h urine sample. 14 oral dose of

C folic acid.

B, 6-24h urine sample, after an Columns were eluted with a

linear gradient of O - 10 M sodium chloride in 0.05 M phosphate buffer pH 7.0 containing 5mg % dithiothreitol. 0 - 0 NaCl gradient III metabolite C I metabolite A II metabolite

B

IV

metabolite

D

416

50

58

66

Sample no. (5 mis) FIGURE ( 3) D.E.A.E. cellulose chromatograms of urine samples from 14 rats with Walker 256 tumours after an oral dose of C folic acid.

A. 0-6h sample, B. 6-24h sample.

Chromatograms were

eluted with a linear gradient of 0-1.0 NaCl in 0.05 M phosphate buffer pH 7.0 containing 5 mg % dithiothreitol. o -o

NaCl gradient.

417

graphic analysis on DEAE cellulose or sephadex G,15.

At the

end of the experiment animals were killed and livers removed (5) for the determination of liver

folates.

Quantitative analysis of urine samples

(Fig.l) showed that

7.2% of the dose was found in the 0.6h. urine sample in normal rats and 5.6% in the 6-24h. sample.

The corresponding

figures for tumour-bearing rats were 7.3% and 12.9%.

Total

recoveries in urine in 48h. were 13.6% in normal animals and 22.0% in tumour-bearing animals.

Thus in both cases the maj-

ority of the urinary radioactivity is excreted in the first 24h. following administration of the dose.

It is apparent

that tumour-bearing rats excrete more of the dose in 48h. than do normal rats, and that the majority of the urinary radioactivity is excreted in the first 6h. in normal in the 6-24h. sample in tumour-bearing rats.

rats,but

Quantitative

analysis of faeces showed that more of the administered radioactivity was excreted in the faeces of normal rats than in tumour-bearing rats

(37.7%).

(44.5%)

Both normal and

tumour-

bearing rats retained about 15% of the dose in the liver after 48h.

About 4% of the dose was found to be retained in

the tumour after two days. Qualitative analysis of 0-6h. and 6-24h. urine samples

(Figs.

2 and 3) showed the presence of four radioactive components in all urine samples examined D).

(designated metabolites A.B.C. and

Metabolite D, eluting at high concentrations of sodium

chloride was found to be chromatographically folic acid.

The quantity of this metabolite in urine decreas-

es with increasing time.

Metabolite C could not be separated

on DEAE cellulose or sephadex G.15 from folic acid folic acid.

identical with

(Fig.4).

5-methyltetrahydro-

Metabolite B was identified as 10 formyl-

Metabolite A eluted from sephadex G.15 close to

the void volume, and on DEAE cellulose at low concentrations of sodium chloride.

This behaviour is identical with that of

authentic unlabelled 4a-hydroxy-5-methyltetrahydrofolic

acid.

4-18

40

44

Sample no (5 mis) Figure (4) Sephadex G.15 chromatogram of peak C from DE52 mixed with unlabelled 5-methyltetrahydrofolic acid. buffer was

The eluent

0.05M sodium phosphate pH 7.0 containing

5 mg % of dithiothreitol. • - • 5 - methyltetrahydrofolic acid o - o radioactivity. Q

9

H

3

N. •ch

H N

h

2

n'

N i ^ N ^

Figure (5)

H-

2

— n h

^ ~ ~ ^ c o - n h - c h

( c h

C O O H

4a-hydroxy-5-methyltetrahydrofolic acid.

2

)

2

c o o h

4-19

Metabolite A did not separate from

4a-hydroxy-5-methyltetra-

hydrofolic acid o n DEAE cellulose or sephadex G.15.

In

a d d i t i o n , no o t h e r folate m o n o g l u t a m a t e s b e h a v e in a

similar

m a n n e r , a n d m e t a b o l i t e A c o u l d b e s e p a r a t e d f r o m all

other

folate monoglutamates that were available. s u g g e s t e d t h a t m e t a b o l i t e A is folic acid

(Fig. 5).

I t is,

therefore,

4a-hydroxy-5-methyltetrahydro-

This metabolite has not previously

b e e n i d e n t i f i e d in r a t urine.

4a-hydroxy-5-methyltetrahydro-

folic a c i d c a n b e f o r m e d f r o m 5 - m e t h y l t e t r a h y d r o f o l i c

acid.

5-methyltetrahydrofolic

a c i d is r e a d i l y o x i d i s e d to

5 , 6 - d i h y d r o f o l i c acid.

This c o m p o u n d c a n t h e n a d d o n w a t e r

to f o r m 4 a - h y d r o x y - 5 - m e t h y l t e t r a h y d r o f o l i c

acid.

5-methyl-

However,

c o n t r o l e x p e r i m e n t s i n d i c a t e t h a t m e t a b o l i t e A is n o t 14 from

C 5-methyltetrahydrofolic acid either during

i o n o f u r i n e s a m p l e s o r in s u b s e q u e n t a n a l y t i c a l It is, t h e r e f o r e , s u g g e s t e d t h a t

formed collect-

procedures.

4a-hydroxy-5-methyltetra-

h y d r o f o l i c a c i d a r i s e s w i t h i n the a n i m a l , e i t h e r b y p r o c e s s e s , or b y a d d i t i o n of w a t e r to folic a c i d , f o r m e d by o x i d a t i o n o f

enzymic

5-methyl-5,6-dihydro-

5-methyltetrahydrofolic

acid. A l t h o u g h all u r i n e s a m p l e s c o n t a i n e d acid,

10-formyltetrahydrofolic

5-methyltetrahydrofolic

acid and

4a-hydroxy-5-methyl-

t e t r a h y d r o f o l i c a c i d the a m o u n t o f e a c h m e t a b o l i t e

altered

w i t h t i m e , a n d t h e r e w a s a d i f f e r e n t d i s t r i b u t i o n of m e t a b o l i t e s b e t w e e n n o r m a l , a n d t u m o u r - b e a r i n g rats T h u s in n o r m a l r a t s 5 - m e t h y l t e t r a h y d r o f o l i c a c i d 20% o f u r i n a r y r a d i o a c t i v i t y 40% in the 6 - 2 4 h . s a m p l e .

(Fig.

6).

represents

in t h e 0 - 6 h . u r i n e s a m p l e

T h e a m o u n t of u r i n a r y

and

radio-

a c t i v i t y t h a t is 4 a - h y d r o x y - 5 - m e t h y l t e t r a h y d r o f o l i c

acid

i n c r e a s e s w i t h i n c r e a s i n g t i m e , w h e r e a s q u a n t i t y o f 10 formyltetrahydrofolic acid decreases.

T h e p a t t e r n of

u r i n a r y m e t a b o l i t e s in t u m o u r - b e a r i n g rats is different.

significantly

In b o t h 0 - 6 h . a n d 6 - 2 4 h . u r i n e s a m p l e s

the

m a j o r u r i n a r y m e t a b o l i t e is 1 0 - f o r m y l t e t r a h y d r o f o l i c a n d the a m o u n t o f this m e t a b o l i t e i n c r e a s e d w i t h

acid,

increasing

4-20

Percentage of urinary

radioactivity

associated with each metabolite

Normal rata

A

B

0-6h.

28.0

30.2

19.9

21.5

6-24h.

38.7

5.9

39.2

8.5

A

B

C

D

Tumour-bearing

rats

C

D

0-6h.

21.7

46.2

22.0

7.5

6-24h.

15.0

56.0

7.7

3.1

Figure (6) Distribution of urinary radioactivity

between

metabolites A, B, C, and D in normal and tumour bearing rats.

421

Sample no. (5 mis) FIGURE (7) A.

Sephadex G.15

chromatogram of a tumour extract.

Eluent buffer, 0.05 M sodium phosphate pH 7.0 containing 5 mg % dithiothreitol. B.

D.E.A.E. cellulose chromatogram of the peak from sephadex G. 15 (above).

Gradient 0 - 2.0 M sodium chloride in 0.05 M

phosphate buffer pH 7.0 containing 5 mg % dithiothreitol. o - o

sodium chloride gradient.

422 time.

The percentage of urinary radioactivity

as 5-methyltetrahydrofolic acid and

appearing

4a-hydroxy-5-methyltetra-

hydrofolic acid in tumour-bearing rats decreased with

increas-

ing time. Qualitative analysis of radioactivity

in livers and tumours

showed that approximately 90% of the radioactivity

eluted

as a single peak immediately after the void volume on sephcdax G.15

(Fig. 7).

Thus it appears that two days after admin14

istration of oral

C folic acid 90% of radioactivity

in the

liver and tumour tissue was a folate of high molecular weight. Other workers

(6) have equated these high molecular weight

folates with folate polyglutamates.

Analysis of liver and

tumour-extracts on DEAE cellulose failed to show the presence of peaks at high tube number normally associated with polyglutamates

(Fig. 7).

In all cases the radioactivity

in liver

and tumours eluted from DEAE cellulose columns in the same position as authentic folate monoglutamates

(5-methyltetra-

hydrofolic acid' or 10 formyltetrahydrofolic acid). In addition the total percentage of the dose excreted in 14 urine as

C folic acid in normal rats is significantly

larger than that excreted in urine of tumour-bearing

rats,

indicating that tumour-bearing rats metabolize a given dose of folic acid faster than normal rats. Much of the work on the metabolism and distribution of folic acid has done of using3H, but H folic acid. Workers do not mention any been exchange in many cases the 3 results obtained with H folic acid are not the same as 14 those obtained with C folic acid. It was, therefore, decided to use a dual-labelled species of folic acid to look at the metabolism of folic acid in normal '.rats.

Animals

(300g body wt) received oral dose of dual labelled folic acid (70 mg/kg, 1 y C i

2 v4C

and 2y Ci 3.5.9

3

H folic acid).

Urine

was collected over 48h as three samples, 0-6h, 6-24h and 2448h.

Faeces were collected as a single sample.

of the experiment livers were removed and treated

At the end (5) to

423 Urines Animal

0-6 h 3

14

H

C

6-•24 1" 3

both

1 4 c both

H

3

H 1 4 c both

3.4

3

H

24.1

14

c:

both

1

5.4

2

8.6 3.6

3.8 17.7 3.9 4.8 7.9 0.9 1.4 34.2 8.4 10.0

3

8.1 3.6

3.9 23.0 6.2 4.9 4.4 1.0 1.2 35.5 9.5 11. 3

4

-

5.8

-

Figure

-

15. 3

Total recovery

24-48 h

-

-

5.1

-

-

-

-

-

0.4

-

-

-

-

-

11. 3

-

(8) Percentage recovery of radioactivity in urine of normal animals over 48h following an oral dose of 3 14 either H folic acid or C folic acid or a mixture of the two.

Figure

(9)

Sample no. (5 mis) D.E.A.E. cellulose chromatograms of urine of animals receiving a mixture of

• -•

th

and o - o

14

C

^H folic acid

folic acid.

14C

x - x sodium chloride

gradient

prevent breakdown of folate polyglutamates by conjugase. Quantitative analysis of urine showed a significant difference 14 3 in the total urinary recovery of C and H.(Fig. 8). Considerably more

^H was recovered in urine in 4 8h. than

"^C .

Qualitative analysis of urine showed that a similar pattern of urinary metabolites as that obtained in previous experiments using 14C folic acid, but the recovery of 14C in each peak eluted from the column was not the same as the recovery 3 14 3 of H. Peaks containing C always contained some H but, 3 on some chromatograms, peaks of H alone were detected. (Fig. 9). DISCUSSION The results reported here demonstrate the presence of a previously unidentified metabolite, 4a-hydroxy-5-methyltetrahydrofolic acid, in rat urine. analytical artifact.

This metabolite is not an

4a-hydroxy-5-methyltetrahydrofolic acid

is not found in the urine of very young rats up to 24h. (6) after an oral dose of folic acid, and appears to be the 14 major urinary metabolite 72h. after an oral dose of C folic acid to mature rats.

Folate polyglutamates are reported

to be formed slowly in the rat liver (7).

Polyglutamates were 14 not found in rat lymphocytes after a dose of 5- C - methyltetrahydrofolic acid, and only 5-methyltetrahydrofolic acid is detected. (8) . It is suggested that 4a-hydroxy-5-methyltetrahydrofolic acid is a breakdown product of retained folates.

Tumour bearing

rats excrete less 5-methyltetrahydrofolic acid and more 10formyltetrahydrofolic acid. This is consistent with a rapid14 ly dividing tissue. More of a dose of C folic acid is channelled into 10-formyltetrahydrofolic acid which is needed 14 as a coenzyme, and less C folic acid is converted to 5methyltetrahydrofolic acid, which acts as a shortterm store of folic acid, and to high molecular weight forms, hence

425 less 4a-hydroxy-5-methyltetrahydrofolic urine.

acid appears in the

Tumour bearing rats excrete more of a dose of folic

acid in urine and less in faeces than normal rats.

This

could be because of enhanced absorption from the gut, or be a result of differing levels of metabolites in the animal, thus tumour-bearing rats excrete less radioactivity the intestine

into

(via the bile) than do normal rats.

The results obtained when dual-labelled

folic acid

was given to normal rats are indicative of exchange of ^H in folic acid, possibly with body water. It is interest3 14 ing to note that the percentage of H and C recovered as folic acid in rat urine were equal. This finding suggests 3 H tritium takes place during metabolism

that exchange of

within the animal, and that tritiated folic acid itself does not'exchange- u n d e r the conditions used in this experiment. The majority of the work on folate polyglutamates has been carried out using tritium tracers,the failure of this

labor-

atory to obtain conclusive evidence for14 polyglutamate formation may be because of the use of a C instead of a tritium tracer. The authors are grateful to the Cancer Research for financial

support.

Campaign

4-26

REFERENCES 1)

Blair,

J.A.

& D r a n s f i e l d , E.

Biochem.J. 907-914

2)

Beavon, J . R . G .

123,

(1971).

Ph.D. T h e s i s , U n i v e r s i t y of Aston (1973).

3)

D r a n s f i e l d , E.

Ph.D. T h e s i s , U n i v e r s i t y of Aston (1972).

4)

Vidal, A . J .

& Stokstad,

E.L.R.Biochim.Biophys.Acta362, 245 -

5)

B i r d , O.D.,McGlohon, J.M.

257.

& V a i t k u s , J.W. Anal. 12, 1 8 - 3 5

6)

Malgani, M.A.K.

7)

Brown, J . P . ,

8)

unpublished

Biochem.

(1965). results.

Davidson, G.E. and S c o t t , J . M . Biochim. bipphys. A c t a . , 343, 7 8 - 8 8 ,

L a v o i e , A . , Tripp, E. & Hoffbrand, A . V . , C l i n . Mol. Med., 4 7 , 6 1 7 - 6 3 0

(1974)

Sei.

(1974).

DISCUSSION Boyle: Were antioxidants routinely incorporated during your workup and chromatographic procedures ? Barford: Antioxidants were present during collection of urine and subsequent analytical procedures. Boyle: You mentioned the 4a-hydroxy compound as a hydration derivative of 5-methyldihydrofolic acid. Could the l a t t e r be the metabolite actually present in the urine ? The 5-methyltetrahydrofolic acid which you have detected could have been formed during workup and chromatography by antioxidant reduction of naturally occurring 5-methyldihydrofolic acid. Barford: 5-Methyldihydrofolic acid could be present in urine, i t would not be detected as such in our experiments. 5-Methyldihydrofolic acid could be produced in the bladder by oxidation of 5-methyl-tetrahydrofolic acid and the 4a-alcohol formed from this. However, we find that the only detectable

4-27 14C -labelled metabolite in urine samples collected 48 h and 72 h after a dose of ^C-folic acid is the 4a-hydroxy compound. Cooper: We have recently published analysis of ^H-folates in human bile after it1s absorption from the gut. The largest fraction of 3 H, appearing within minutes of feeding chromatographed in the same position as your material, and did not support growth of microorganisms. It may be the same material. Barford:

Yes, we agree.

Rowe: This question is directed not only to Dr. Barford but also to any other members of the audience some of whom have had considerable experience with 3'5',9-^H-folic acid. - Has anyone any idea of the nature of tritium exchange postulated as the reason for the differential isotope recovery in the double label experiments ? Barford: 315' -folic acid as supplied by the radiochemical center Amnersham, U.K., has about 30 % of the tritium in the 9-position. The ratio of 3H to 14c in the urinary folic acid is approx. the same as the ratio of 3 H to 14c in the administered folic acid. This observation suggests that 3 h exchange may be taking place during metabolism of the folic acid. It has been shown that folic acid reacts in deuterium oxide - deuteriotrifluoroacetic acid mixtures to form 3",5-deuterio folic acid (Rosenberg, Palladino, Hackey & Blair - unpublished). Albert: To which atoms in the folic acid derivative were the tritium atoms attached, to carbons or to exchanging atoms ? Barford:

The tritium atoms are all attached to carbon atoms,

Scrimgeour: Refering again to the properties of coenzyme oxidation, I am surprised that you found 10-formyl tetrahydrofolate, but folate and not tetrahydrofolate itself. The rates of oxidation of these compounds are identical. Barford: We think that the folic acid found in urine is excreted as such and is not produced by oxidation of tetrahydrofolic acid. The amount of folic acid appearing in urine decreases with increasing time, and a higher dose of folic acid results in the appearence of more folic acid in urine. The principal degradation products of tetrahydrofolic acid are xanthopterin and 2-amino-4-hydroxypteridine, and they are not found. Taylor: Could you, or can anyone else present, summarize for me the structural evidence supporting the assigned structure for 4a-hydroxy-5-methyltetrahydrofolic acid ? Barford: 4a-Hydroxy-5-methyl tetrahydrofolic acid was synthesized chemically by the method of Gapski, Whiteley & Huennekens, Biochemistry 10, 293o, (1971). This paper describes the method of structural assignment.

"Prune"/"Killer-of-Prune": A Complementary Lethal System in Drosophila Melanogaster Affecting Pteridine Metabolism J.H.P. Hackstein

The recessive sex-linked, gene "prune" ("pn", 1-0.8) is known to affect eye colour and to form a system of complementary lethality with the autosomal dominant gene "Killer-of-prune" ("K-pn", 3 —102.9) (1> 2). Every individual which carries one or two doses of the "K-pn" gene and which is likewise homozygous or hemizygous for "pn" dies during larval development as either a 2n