Chemistry and Biology of Pteridines: 7 St. Andrews, Scotland, September 21–24, 1982 [Reprint 2014 ed.] 9783111619453, 9783110085600

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Chemistry and Biology of Pteridines Pteridines and Folic Acid Derivatives

Chemistry and Biology of Pteridines Pteridines and Folic Acid Derivatives Proceedings of the Seventh International Symposium on Pteridines and Folic Acid Derivatives Chemical, Biological and Clinical Aspects St. Andrews, Scotland, September 21-24,1982 Editor John A. Blair

W DE Walter de Gruyter • Berlin • New York 1983

Editor John A. Blair, B.Sc., Ph.D., D.Sc., C.Chem.,F.R.S.C. Professor of Chemistry The University of Aston in Birmingham Department of Chemistry Gosta Green, Birmingham B4 7ET Great Britain

Library of Congress Cataloging in Publication

Data

International Symposium on Pteridines and Folic Acid Derivatives: Chemical, Biological, and Clinical Aspects (7th: St. Andrews, Scot.) Chemistry and biology of pteridines. Includes bibliographies and indexes. Congresses. I. Blair, John A., 1927. II. Title. [DNLM: 1. Folic acid-Methabolism-Congresses. 2. Pteridines-Congresses. W3 IN922j 7th 1982 c / QU18816161982c] QP801.P691585 1982 599'.01926 ISBN 3-11-008560-7

CIP-Kurztitelaufnahme

der Deutschen

83-7666

Bibliothek

Chemistry and biology of pteridines. - Berlin; New York; de Gruyter Pteridines and folic acid derivatives: proceedings of the 7. Internat. Symposium on Pteridines and Folic Acid Derivatives, Chem., Biolog. and Clin. Aspects, St. Andrews, Scotland, September 21-24,1982 / ed. John A. Blair. Berlin; New York: de Gruyter, 1983. (Chemistry and biology of pteridines) ISBN 3-11-008560-7 NE: Blair, John A. [Hrsg.] ; International Symposium on Pteridines and Folic Acid Derivatives, Chemical, Biological and Clinical Aspect (1982, Saint Andrews). Copyright © 1983 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: Karl Gerike, Berlin. - Binding: Dieter Mikolai, Berlin. - Printed in Germany.

Preface This book records the proceedings of the Seventh International Symposium on Pteridines and Folic Acid Derivatives held in St. Andrews, Scotland from 21st to 24th September, 1982 at which 274 participants from 24 countries were present. As with the previous symposia a wide range of topics was presented. This symposium had much larger coverage of tetrahydrobiopterin chemistry and biochemistry than previously, reflecting the present considerable upsurge of interest. It was also apparent that there is great increase in the sophistication of studies in the areas represented at the symposium, a trend to be greatly welcomed. In organising the symposium and thus the general structure of this book, leading scientists were invited to give keynote lectures summarising recent developments in their field of expertise. These are to be found at pages 1, 23, 93, 131, 197, 263 and 321. It is hoped they will be of value to the reader who wishes to inform himself of developments outside his own special interests. The many contributions from other authors describe the most recent developments in research in the chemistry and biochemistry of pteridines. By putting both these aspects together this volume should provide a detailed survey of the present state of these fascinating and important fields of study. As an experiment brief reports of the discussions and papers presented at each session have been included. Comments on this procedure will be welcomed. The next symposium scheduled for 1986 in Canada should bring forth a further crop of new and fascinating studies. John A. Blair Birmingham, April 1983

ACKNOWLEDGEMENTS

The organisers gratefully acknowledge financial and other assistance from the following

organisations:

The British Petroleum Company Limited Burroughs Wellcome Co U.S.A. Glaxo Holdings Limited Imperial Chemical Industries Limited (Pharmaceuticals Division) Merck Sharp and Dohme Limited Pfizer Central Research Roche Products Limited Shell U.K. Limited Smith, Kline and French Laboratories The Wellcome Foundation Limited

0 Cancer Research Campaign Scottish Home and Health Department Scottish International Education Trust University of St. Andrews University of

Strathclyde

PARTICIPANTS # = Accompanying Guest * = Steward/Secretary ABOU-DONIA, M.

BOYLE, P.H.

ABOU-HADEED, K.

BRAVERMAN, E.B.

AMY, N.K.

BROOM, A.D.

#AMY, C.M.

#BROOM, M.J.

ANGERBAUER, R.

BROWN, D.J.

ARMAREGO, W.L.F.

#BROWN, J.C.

AYLING, J.E.

BROWN, G.M.

AYRES, B.E.

BUCHANAN, J.

BACCANARI, D.P.

CALVERT, H.

#BACCANARI, F.

CHANARIN, I.

BACHER, A.

CHARLTON, P.A.

BAILEY, S.W.

CICHOWICZ, D.J.

BARFORD, P.A. BAXTER, A.

#CICHOWICZ, M.B CLAASSEN, V.P.

BEARDSLEY, G.P.

CODY, V.

#BEARDSLEY, D.

COOK, R.J.

BEDDELL, C.R.

COOPER, B.A.

BELLAHSENE, Z.

CORROCHER, R.

BENKOVIC, S.J.

COSSINS, E.A.

#BENKOVIC, P.A.

DA COSTA, M.P.

BERRY, H.K.

COTTON, R.G.H.

BERTINO, J.R.

COWARD, J.K.

#BERTINO, M.P.

COWARD, M.

BEVAN, A.W.

CROSTI, P.

BIANCHETTI, R. BILLINGS, R.E.

CURTIUS, H.C. DANIELS, A.J.

BIRDSALL, B.

DEACON, R.

BLAIR, J.A.

DEGRAW, J.I.

BLAKLEY, R.L.

#DEGRAW, R.J.

#BLAKLEY, B.E.

DEV, I.

BOCK, L.

#DEV, M.

BOGNAR, A. L.

DHONDT, J.L.

X

DIDDENS, H.

HASEGAWA, H.

DOIG, M.T.

HAWKES, T.R.

DONLON, J.C.

HECKEL, A.

DÜCH, D.S.

HENDERSON, G.B.

#DUCHr K.

HENKIN, J.

DUNLAP, R.B.

HOLM, J.

DWIVEDI, M.C.

HÖRNE, D.W.

EGGAR, C.S.

HUEBSCH, W.

ERBE, R.W.

HUENNEKENS, F.M.

#ERBE, E.T.

#HUENNEKENS, B.L

#ERBE, J.R.

IMAIZUMI, S.

#ERBE, J.A.

JACKMAN, A.L.

FERONE, R.

JACKSON, R.C.

FERRE, J.J.

JACOBSON, K.B.

#GOMEZ, M.D.M. FINK, M.

•JACOBSON, P.

FINK, R.

JANNE, K.

FIRGAIRA, F.A.

JENSEN, J.K.

#FIRGAIRA, A.

JOHNS, D.G.

FREISHEIM, J.H.

JOHNSON, J.L.

#JACOBSON, D.

IFREISHEIM, S.J.

JONES, R.C.F.

FUCHS, D.

JONES, T.R.

GALIVAN, J.H.

JONGEJAN, J.A.

GEDDES, A.J.

#JONGEJAN, K.S.

GHISLA, S.

JOULE, J.A.

GLOWKA, E. GOODFORD, P.J.

KAPATOS, G.

GOTO, M. GREADY, J.E.

KAUFMAN, S.

GROSSOWICZ, N.

KEATING, S.

GUEST, A.A.E.

KELLY, M.

HAAVIK, J.

KEMPTON, R.J.

KATOH, S. KAUFMAN, E.

HAMRELL, M.R.

#BLACK, A.M.

HANSEN, S.I.

KIENZLE, F.

HARDING, N.G.L.

KIRIASIS, L.

IHARDING, J.W. HARZER, G.

KISLIUK, R.L. KNIGHT, C.H.

KOHASHI, M. ROMPIS, I. KOZLOFF, L.M. #KOZLOFF, J.B. KUYPER, L.F. LAZARUS, R.A. LEACH, M.J. LEE, J.W. LEEMING, R.J. LEVINE, R.A. LEWIS, G.P. #LEWIS, A.K. LOCKHART, R.J. LOIDL, P. LONG, M. LOVE, B.E. LYKKELUND, C. MAGER, H.I.X. MALEY, F. MALEY, G.R. MANGUM, J.H. MARK, J.E. MASADA, M. MATHEWS, C.K. #MATHEWS, C.Z. MATTHEWS, D.A. MATTHEWS, R. G. MATSUURA, S. DE MEESTER, J.W.G. MILLER, A.A. MILSTEIN, S. #MLADENOVIC, J. MOHYUDDIN, F. #MOHYUDDIN, S. MOLLOY, A. McGUIRE, J. McMARTIN, K.E.

*McROBERTS, A. NAIR, M.G. NICHOL, C.A. #NICOL, R.H. NIEDERWIESER, A. NIXON, J.C. NORONHA, J.M. O'BROIN, S.D. O'DONNELL, R.A. O'MAHONY, M.J. 01MURCHU, C. 0'SULLIVAN, H. PARNIAK, M.A. IPARNIAK, J. PARVEEN, H. PATON, D. PENDERGAST, W. PERRY, J.R. PETERSON, D.L. PFLEIDERER, W. PHEASANT, A.E. PIEPER, H. PIPER, W.N. #PIPER, M.K. PRIEST, D.G. RABINOWITZ, J.G. RAJAGOPALAN, K.V RAMESH, K.S. #SANDHYA, K.R. RAO, D.N. REIBNEGGER, G. REYNOLDS, E.H. »ROBERTS, C. ROBERTS, G.C.K. *ROBINSON, D.I. RODE, W. ROKOS, H.

XII

ROKOS, K.

SUCKLING, C.J.

ROOD, R.

*SUGDEN, P.

#ROOD, L.

SUGIMOTO, T.

ROSENBLATT, D.S.

TAYLOR, E.C.

ROSOWSKY, A.

#TAYLOR, V.C.

#ROSOWSKY, E.

THEN, R.L.

ROTH, B.

#THEN, R.M.

ROTHENBURG, S.

TILKIAN, S.

ROWE, P.B.

#TILKIAN, R.

SABB, A.L.

TISLER, M.

SAUER, H.

TSUSE, M.

SCHALHORN, A.

*VALENTE, E.

SCHIRCH, L.V.

VISSER, K.

SCHWALBE, C.H.

VIVEROS, H.O.

SCHWINCK, I.

WAGNER, C.

SCOTT, J.M.

#WAGNER, M.J.

SEARLE, F.F.

WATKINS, D.

SHANE, B.

WAXMAN, S.

MANDELBAUM-SHAVIT, F.

WEBER, R.O.

*SHEPHERD, T.

WEIR, D.G.

SHERWOOD, R.F.

WELSH, W.J.

SILVA, F.J.

WHITBURN, S.B.

SIMAY, A.

WHITEHEAD, V.M

SMITH, D.M.

#WHITEHEAD, S.

*SMITH, M.

WHITELEY, J.

SRINIVASAN, A.

WEIDERRECHT, G

STAMMERS, D.K.

WILSON, L.

STEINBERG, S.E.

de WIT, R.

STOKSTAD, E.L.R.

WOOD, H.C.S.

ISTOKSTAD, E.

#WOOD, J.D.

STONE, D.

WRIGGLESWORTH,

STRAMENTINOLI, G.

YANAGISWA, R.

STRUM, W.B.

YOUNG, D.W.

#STRUM, F.

ZIEGLER, I.

Contents

The G o w l a n d H o p k i n s

Lecture:

T r a n s p o r t of F o l a t e a n d P t e r i n

Compounds

F.M. Huennekens, M.R. Suresh, C.E. Grimshaw, D.W. Jacobsen, E.T. Quadros, K.S. Vitols, G.B. Henderson

Chemical Aspects

1

(1)

Scottish International Education Trust

Lecture:

S y n t h e t i c M e t h o d s in P t e r i d i n e C h e m i s t r y : Some A p p l i c a t i o n s to P t e r i d i n e N a t u r a l P r o d u c t s E.C. Taylor

23

Synthesis, Chemical and Enzymatic Properties, and T h e r a p e u t i c P o t e n t i a l of 6 , 6 - D i s u b s t i t u t e d T e t r a h y d r o and Quinoid-Dihydro-Pterins S.W. B a i l e y , J.E. A y l i n g

51

T h e S t r u c t u r e of Q u i n o n o i d D i h y d r o p t e r i n s [2-Amino-7,8(6H)-Dihydropteridin-4-ones] W . L . F . A r m a r e g o , P. W a r i n g

Some S i m p l e

57

s-Triazolopteridines

D . J . B r o w n , K. S h i n o z u k a

63

XIV L i n e a r T r i c y c l i c A n a l o g s of

Pteridines

W. P e n d e r g a s t , J . H . C h a n

69

R e a c t i o n s of F u r a z a n o [ 3 , 4 - d j P y r i m i d i n e s a n d t h e i r A p p l i c a t i o n to P t e r i d i n e S y n t h e s i s P.H. Boyle, R.J. Lockhart

S y n t h e s e s of some B l o c k e d

73

7,8-Dihydropteridines

R.C. C a m e r o n , W . J . S . L y a l l , S.H. N i c h o l s o n , D.R. Robinson, C.J. Suckling, H.C.S. W o o d

S y n t h e s i s of C h i r a l R e d u c e d P t e r i d i n e

79

Coenzymes

P. D o y l e , A. S z a k o l c a i , D . W . Y o u n g

Report on

85

Discussions

S.J. B e n k o v i c

91

Chemical Aspects

R e a c t i v i t y of

(2)

Pteridines

W . P f l e i d e r e r , R. B a u r , M . B a r t k e , H. L u t z

S y n t h e s i s a n d A n t i t u m o r A c t i v i t y of

93

10-Deazaminopterins

J . I . D e G r a w , V . H . B r o w n , H. T a g a w a , R.L. Y. G a u m o n t , F . M . S i r o t n a k

Kisliuk,

109

S y n t h e s i s a n d B i o l o g i c a l A c t i v i t y of 5 - D e a z a f o l i c A c i d and 5-Deazaaminopterin E . C . T a y l o r , C. T s e n g , P . J . H a r r i n g t o n , G.P. A. R o s o w s k y , M. W i c k

Beardsley,

115

XV

The Nor-Analogues of Folic Acid M.G. Nair, M.K. Rozmyslovicz, R.L. Kisluik, F.M. Sirotnak, Y. Gaumont

121

Report on Discussions B. Roth

127

Neurochemical and Clinical Aspects

Metabolism of Pteridine Cofactors in Neurochemistry C.A. Nichol, O.H. Viveros, D.S. Duch, M.M. Abou-Donia, G.K. Smith

131

Pteridines in Cancer and Other Diseases K. Rokos, H. Rokos, H. Frisius, M. Hüfner

153

Dihydropteridine Reductase Activity in Human Breast Cancer. Studies of 580 Cases and Correlations with Hormone Receptors and Cellular Typing J.L. Dhondt, C. Herlin, J. Bonneterre, M.C. Vie, J. Lefebvre, A. Demaille

159

A Critical Appraisal of Methods for the Quantitative Analysis of Tetrahydrobiopterin, Dihydrobiopterin and Biopterin in Human Urine, Serum and CSF J.A. Blair, S.B. Whitburn, A.E. Pheasant, R.J. Leeming, C. Morar, A. Al-Beir

165

Inhibition of Pterin Biosynthesis in the Adrenergic Neuroblastoma N1E115 by Tetrahydrobiopterin and Folate G. Kapatos, S. Kaufman

171

XVI

Penetration of Reduced Pterins into Rat Brain: Effect on Biogenic Amine Synthesis R.A. Levine, W. Lovenberg, A. Niederwieser, W. Leimbacher, U. Redweik, H. Staudenmann, H.-Ch. Curtius 177

Neopterin Deficiency (GTP Cyclohydrolase I Deficiency), a New Variant of Hyperphenylalaninemia A. Niederwieser, W. Staudenmann, M. Wang, H.-Ch. Curtius, M. Atares, J. Cardesa-Garcia

183

Folate Pathways in Cells from Fragile X Syndrome Patients and Carriers R.W. Erbe, J.C. Wang

189

Report on Discussions R.G.H. Cotton

195

Enzymology

The Interaction of Substrates and Inhibitors with Dihydrofolate Reductase G.C.K. Roberts

197

Catalytic Site of Dihydrofolate Reductase R.L. Blakley, L. Cocco, J.A. Montgomery, C. Temple Jr., B. Roth, S. Daluge, R.E. London

215

Structure, Function and Affinity Labelling Studies of Dihydrofolate Reductase J.H. Freisheim, S.S. Susten, T.J. Delcamp, A. Rosowsky, J.E. Wright, R.J. Kempton, D.T. Blankenship, P.L. Smith, A.A. Kumar

223

XVII

Methylene Tetrahydrofolate Reductase: Studies in a Human Mutant and Mammalian Liver G.P. Lewis, P.B. Rowe

229

Studies on Methylenetetrahydrofolate Reductase from Pig Liver: Catalytic Mechanism and Regulation by Adenosylmethionine M.A. Vanoni, S.C. Daubner, D.P. Ballou, R.G. Matthews .... 235

Role of Methylenetetrahydrofolic Acid Reductase in Regulation of Folic Acid Metabolism and its Relation to the Methyl Trap Hypothesis E.L.R. Stokstad, M. M.-S. Chan, J.E. Watson

241

The Mechanism of Action of the Transformylase Enzymes G.K. Smith, W.T. Mueller, P.A. Benkovic, L.J. Slieker, C.W. DeBrosse, S.J. Benkovic

247

Folypolyglutamate Synthesis in Neurospora Crassa E.A. Cossins, P.Y. Chan

251

Report on Discussions D.P. Baccanari, L.F. Kuyper

257

Clinical Aspects: Cancer

Folates and Cancer Chemotherapy J.R. Bertino, J.J. McGuire ....

263

XVIII

Inability to Increase the Mobilisation of Liver Folates During Folate Deficiency Suggesting that this and Other Organs do not act as a Potential Folate Store for Marrow and Other Rapidly Proliferating Cells J.M. Scott, A. Molloy, A. Smithwick, P. McGing, D.G. Weir

275

A High Molecular Weight Nonfunctional Immunoreactive Form of Dihydrofolate Reductase in L1210 Leukaemia Cells S.P. Rothenberg, M.P. Iqbal

281

Further Characterization of the Pterdine Binding Variant of Alphas-Acid Glycoprotein, Accumulated in the Blood of Patients with Malignant Diseases I. Ziegler, W. Armarego, M. Fink

287

Structure-Activity Relationships among 5-Substituted Lipophilic Diaminopyrimidine Antineoplastic Antifolates V. Cody, E. DeJarnette, S.F. Zakrzewski

293

Attempts to Design Inhibitors of Dihydrofolate Reductase Using Interactive Computer Graphics with Real Time Energy Calculations E.A. Potterton, A.J. Geddes, A.C.T. North

299

Control of Methotrexate Polyglutamate Synthesis in Cultured Rat Hepatoma Cells J. Galivan, M. Balinska

305

Differential Effects of Methotrexate or Fluorodeoxyuridine upon Mitochondrial and Cellular Nucleotide Pools R.K. Bestwick, G.L. Moffett, C. Spiro, C.K. Mathews

311

Report on Discussions J.M. Whiteley

317

XIX Bioorganic Chemistry and Enzymology

C h e m i c a l M e c h a n i s m s in the A c t i o n of P t e r i d i n e

Coenzymes

D.W. Young

321

I r r e v e r s i b l e I n a c t i v a t i o n of R a t L i v e r P h e n y l a l a n i n e Hydroxylase by Reaction with (6S)-L-Erythro-Tetrahydrobiopterin M . P a r n i a k , S. K a u f m a n

345

p-Azidophenylalanine: A Potential Photoaffinity for R a t - L i v e r P h e n y l a l a n i n e H y d r o x y l a s e

Probe

S. W e b b e r , J . M . W h i t e l e y

351

P y r i m i d i n e M o d e l s for the C o f a c t o r of P h e n y l a l a n i n e H y d r o x y l a s e . A u t o x i d a t i o n of 2 , 4 , 5 , - T r i a m i n o - 6 - H y d r o x y pyrimidines J.A. Jongejan, H.I.X. Mager, W. Berends

A c t i v a t i o n of M o l e c u l a r O x y g e n b y Monooxygenases

357

Tetrahydropterin

J.E. A y l i n g , S.W. B a i l e y

363

Mechanistic Studies on Phenylalanine Hydroxylase: S t r u c t u r a l D e t e r m i n a t i o n of the T e t r a h y d r o p t e r i n Intermediates R . A . L a z a r u s , C . W . D e B r o s s e , S.J. B e n k o v i c

P r o p e r t i e s of T h y m i d y l a t e S y n t h a s e f r o m Faecium

369

Streptococcus

R.L. K i s l i u k , K.N. R a o , Y. G a u m o n t , R. D e s c h e n e s , K. V e s t a l , M . K a r a s i k , G . F . M a l e y , M . G . N a i r , C . M . B a u g h

. 375

XX Cloning the Gene for Eukaryotic C^-THF Synthase C. Stäben, J.C. Rabinowitz

381

Report on Discussions W.L.F. Armarego

387

Biochemistry and Biology

The Enzymatic Synthesis of the Drosopterins: Identification of a Pyrimidodiazepine as an Intermediate G.J. Wiederrecht, D.R. Paton, G.M. Brown

391

The Common Precursor of Sepiapterin and Drosopterin in Drosophila: Enzymatic and Chemical Synthesis W.G. Hearl, D. Dorsett, K.B. Jacobson

397

The Role of Pteridines in Regulation of Growth H. Wächter, D. Fuchs, A. Hausen, G. Reibnegger

403

The Regulation of Molybdenum Cofactor in Escherichia Coli N.K. Amy, J.B. Miller

409

Evidence for a Folate Bound to Rat Hepatic Uroporphyrinogen III Cosynthase and its Role in the Biosynthesis of Heme W.N. Piper, J. Tse, R.P. Clement, M. Kohashi

415

Report on Discussions P.H. Boyle

421

XXI Presented

Papers

A e r o b i c O x i d a t i o n of

5,6,7,8-Tetrahydroneopterin

W . L . F . A r m a r e g o , D. R a n d i e s

423

T h e C o n f o r m a t i o n s of 7,8 ( 6 H ) - D i h y d r o p t e r i n S u b s t r a t e s a n d the A c t i v i t y of D i h y d r o p t e r i d i n e R e d u c t a s e (E.C.1.6.99.10) W . L . F . A r m a r e g o , P. W a r i n g

429

Dihydrofolate Reductase: The Stereochemistry Inhibitor Selectivity

of

D . A . M a t t h e w s , J . T . B o l i n , D . J . F i l m a n , K.W. J. K r a u t

Volz,

S y n t h e s i s of an 8 - D e a z a A n a l o g of the I n t e r m e d i a t e the T h y m i d y l a t e S y n t h e t a s e R e a c t i o n (1)

435

in

A . D . B r o o m , A. S r i n i v a s a n

445

T h e S y n t h e s i s of A n a l o g s of P t e r o y l g l a t a m y l - y - P h o s p h a t e as P o t e n t i a l I n h i b i t o r s of F o l y l p o l y g l u t a m a t e S y n t h e t a s e K.C. Tang, J.K. Coward

S y n t h e s i s a n d A c t i v i t y of

451

8,1O-Dideazaminopterin

J . I . D e G r a w , L . F . K e l l y , R . L . K i s l i u k , Y. F.M. Sirotnak

Gaumont,

457

C N D O / 2 M o l e c u l a r O r b i t a l C a l c u l a t i o n s o n the A n t i f o l a t e D A M P a n d S o m e of its A n a l o g u e s : C o n f o r m a t i o n a l Characteristics W . J . W e l s h , J.E. M a r k , V. C o d y , S.F. Z a k r z e w s k i

463

XXII F l u o r e s c e n t A n a l o g u e s of M e t h o t r e x a t e as P r o b e s Folate Antagonist Molecular Receptors J.H. Freisheim, A.A. Kumar, A.M. Black, G.M. R.J. K e m p t o n , S.S. S u s t e n

for

Anstead,

469

Dihydrofolate Reductase Overproduction Identified by Flow Cytometry Using a Fluorescent Methotrexate Analogue A. R o s o w s k y , J . E . W r i g h t , G.P. B e a r d s l e y , H . M . S h a p i r o

N e w S y n t h e s e s a n d T r a n s f o r m a t i o n s of S o m e and Pteridine 3-0xides

... 475

Pteridines

M . K o c e v a r , B. S t a n o v n i k , M . T i s l e r

P h o t o o x i d a t i o n of

481

Mercaptopteridines

A . H e c k e l , W . Pf l e i d e r e r

R e a c t i v i t y of 6 , 7 - D i c h l o r o - 1 ,

487

3-Dimethyllumazine

K. A b o u - H a d e e d , W . P f l e i d e r e r

493

S y n t h e s i s , R e a c t i v i t y a n d P r o p e r t i e s of 2-Thio- and 2,4-Dithiolumazines

8-Substituted

W. H ü b s c h , W. P f l e i d e r e r

499

The A c t i v i t y of S u l f o n a m i d e - S u b s t i t u t e d B e n z y l p y r i m i d i n e s Against Dihydropteroate Synthase, Dihydrofolate Reductase, and Bacterial Cell Cultures R . M . H y d e , R.A. P a t e r s o n , C . R . B e d d e l l , J . N . C h a m p n e s s , D . K . S t a m m e r s , D . J . B a k e r , P.J. G o o d f o r d , L . F . K u y p e r , R. F e r o n e , B. R o t h , L . P . E l w e l l

S t r u c t u r a l S t u d i e s of A n t i f o l a t e C.H. Schwalbe, V. Cody

505

Drugs 511

XXIII

Isolation and Characterization of a Novel Folate Antagonist G. Schlingmann, E.L.R. Stokstad

517

Synthesis and Biological Properties of the S-8 Analog of 7,8-Dihydropteridinealcohol K. Visser, J.K. Seydel

523

Synthesis of Dihydrobiopterin from Dihydroneopterin Triphosphate in Rat Tissues and Human Blood Corpuscles: Diagnosis of Atypical Phenylketonuria due to Biopterin Deficiency by Assay of an Enzyme Involved in the Synthesis M. Akino, S. Yoshioka, M. Masada, K. Inoue, T. Yoshida, T. Mizokami, N. Matsuo

529

Stereospecific Enzyme Mediated Syntheses of Tetrahydrofolate Derivatives L. Rees, C.J. Suckling, E. Valente, H.C.S. Wood

533

Immobilization of Dihydrofolate Reductase from Amethopterin Resistant Lactobacillus Casei F. Ahmed, R.B. Dunlap

539

X-Ray Studies of the Binding of Trimethoprim, Methotrexate, Pyrimethamine and Two Trimethoprim Analogues to Bacterial Dihydrofolate Reductase D.J. Baker, C.R. Beddell, J.N. Champness, P.J. Goodford, F.E. Norrington, B. Roth, D.K. Stammers

545

A New Spectrophotometry Method for the Assessment of Tight Binding Benzyldiaminopyrimidine Inhibitors of Dihydrofolate Reductase J.G. Dann

551

XXIV T w o I n t e r c o n v e r t i n g C o n f o r m e r s of a D r u g - R e c e p t o r C o m p l e x : The Lactobacillus Casei Dihydrofolate Reductase-NADP+Trimethoprim Complex A . W . B e v a n , B. B i r d s a l l , G.C.K. R o b e r t s , J. A. Gronenborn, G.M. Clore, A.S.V. Burgen

Feeney,

Multiple Conformations Folate-NADP+ Complex

Reductase-

of t h e D i h y d r o f o l a t e

B. B i r d s a l l , A. G r o n e n b o r n , G.M. C l o r e , E.I. G . C . K . R o b e r t s , J. F e e n e y , A . S . V . B u r g e n

The Three-Dimensional folate Reductase

557

Hyde, 563

Structure of M o u s e L1210

Dihydro-

D.K. Stammers, J.N. Champness, J.G. Dann, C.R. Beddell

I s o l a t i o n a n d A m i n o A c i d S e q u e n c e S t u d i e s of folate Reductase from Neisseria Gonorrhoeae

...

567

Dihydro-

D. S t o n e , S.J. P a t e r s o n , R. T a n s i k , D.P. B a c c a n a r i

573

P u r i f i c a t i o n of 7 , 8 - D i h y d r o p t e r o a t e - S y n t h e t a s e f r o m E. Coli by A f f i n i t y - and H y d r o p h o b i c I n t e r a c t i o n Chromatography R. B a r t e l s , L . B o c k

579

I s o l a t i o n a n d C h a r a c t e r i z a t i o n of a n E . C o l i M u t a n t A f f e c t e d in D i h y d r o f o l a t e - a n d F o l y l p o l y g l u t a m a t e Synthetase R. F e r o n e ,

S.C. Singer, M.H. Hanlon,

S. R o l a n d

585

P u r i f i c a t i o n a n d P r o p e r t i e s of 5 - F o r m y l t e t r a h y d r o f o l a t e Cyclodehydrase from Rabbit Liver L. S c h i r c h ,

S.E. Hopkins

Structural Studies on Formyl-Methenyl-Methylene hydrofolate Synthetase from Rabbit Liver D. P e t e r s o n , L . S c h i r c h

591

Tetra597

XXV Utilization of Folate Polyglutamate for Growth by Lactobacillus Casei M. Maclntyre, B.A. Cooper, H. Lue-Shing

603

Folate and Folate Enzymes as Structural Components of Bacteriophage Particles L.M. Kozloff, D. Sadewasser, M. Lute

609

Studies on the Substrate Specificity of Mammalian Folylpolyglutamate Synthetase J.J. McGuire, P. Hsieh, J.R. Bertino, J.K. Coward

615

Properties of Corynebacterium Species Dihydrofolate Synthetase-Folylpolyglutamate Synthetase B. Shane

621

Purification and Characterization of Folylpolyglutamate Synthetase from Lactobacillus Casei and Hog Liver A.L. Bognar, D.J. Cichowicz, B. Shane

627

The Molybdenum Cofactor from Xanthine Oxidase: Quantitative Assay of the Molybdenum Cofactor by Activation of Nitrate Reductase Activity in Extracts of the Nit-1 Mutant of Neurospora crassa T.R. Hawkes, R.C. Bray

633

Structural Studies of Calf Thymus Thymidylate Synthase C.M. Dwivedi, R.L. Kusliuk, G.F. Maley

639

A Comparison of the T-Even Phage Induced and Escherichia Coli Thymidylate Synthases G.F. Maley, M. 'Belfort, F. Maley

645

XXVI

Thermophilicity of Dihydrofolate Reductase and Thymidylate Synthetase from a Thermophilic Microorganism F. Mandelbaum-Shavit, J. Reizer, N. Grossowicz

651

Chemical Characterization of Two Folate-Binding Proteins from Mitochondria: Dimethylglycine Dehydrogenase and Sarcosine Dehydrogenase R.J. Cook, C. Wagner

Allosteric Mung Bean (Vigna radiata) Hydroxylmethyl-Transferase

657

Serine

D.N. Rao, N.A. Rao

663

Comparative Study of the Eye Colour Mutants of Drosophila Melanogaster: Quantification of the Eye-Pigments and Related Metabolites J. Ferré, F. Silva, M.D. Real, J.L. Ménsua

669

Studies on Sepialumazine, the Characteristic Larval Integument Pigment of the Bombyx Mori Kiuki Mutant M. Tsusue, T. Mazda, S. Sakate

675

Macromolecular Regulation of Sepiapterin and Drosopterin Synthesis in the Purple Mutant of Drosophila K.B. Jacobson, J.J. Yim, C.R. Wobbe

681

The Hydroxylation of Phenylpropanoids by Euglena gracilis: Role of Pteridines R. Fink, E. Paur, E.F. Elstner

687

Studies with Old Yellow Enzyme, Reconstitution with Lumazine Analogs as Coenzyme G. Wetzel, S. Ghisla

693

XXVII B i o s y n t h e s i s o f R i b o f l a v i n . O r i g i n of t h e X y l e n e A . B a c h e r , Q . L e V a n , M . Biihler, P . J . H.G. Floss

Ring

Keller,

699

B i o s y n t h e s i s of R i b o f l a v i n . S y n t h e s i s of t h e S u b s t r a t e and of Substrate A n a l o g u e s for P y r i m i d i n e D e a m i n a s e P. N i e l s e n , A . B a c h e r

M e t a b o l i s m a n d F u n c t i o n of P t e r i n C o m p o u n d s Germination

705

in

Seed

M . K o h a s h i , K. T o m i t a , K . I w a i

Thymidylate Synthetase During Proliferation D i f f e r e n t i a t i o n of P h y s a r u m p o l y c e p h a l u m

711

and

P . G r o b n e r , P. L o i d l

I n f l u e n c e o f V i t a m i n B-J2 o n F o l a t e M e t a b o l i s m Lactobacillus leichmannii

717

in

C.P.P. Nair, J.M. Noronha

H i g h - A f f i n i t y B i n d i n g of F o l a t e t o a S m a l l a n d Molecular Size Protein from Human Milk

723

Large

S.I. H a n s e n , J. H o l m , J. L y n g b y e

729

H i g h - A f f i n i t y F o l a t e B i n d i n g in L e u k o c y t e s from N o r m a l Subjects Displays Positive Cooperativity, and the Affinity Depends o n the P r o t e i n C o n c e n t r a t i o n J. Holm, S.I. H a n s e n , J. L y n g b y e

735

C h a r a c t e r i s t i c s of 5 - M e t h y l t e t r a h y d r o f o l a t e Binding to the Folate-Binding Protein from Cow's Whey J. L y n g b y e ,

S.I. H a n s e n , J. H o l m

741

XXVIII

Interactions of Folate-Dependent Enzymes of DNA Precursor Metabolism in T4 Phage-Infected Bacteria J.R. Allen, J.W. Booth, D.A. Goldman, G.W. Lasser, S. Purohit, R.G. Sargent, C.K. Mathews

747

The Regulation of Biopterin Biosynthesis in the Rat S. Milstien, S. Kaufman

753

Biosynthesis of Tetrahydrobiopterin in Mammalian Tissues by De Novo and Salvage Pathways C. A. Nichol, G.K. Smith, D.S. Duch

759

Perspectives on Tetrahydrobiopterin Biosynthesis in Mammals H.-Ch. Curtius, M. Hausermann, D. Heintel, A. Niederwieser, R.A. Levine

765

Human Dihydropteridine Reductase (DHPR) Deficiency F.A. Firgaira, R.G.H. Cotton, D.M. Danks

771

Coordinate Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin in Adrenal Medullary Chromaffin Cells M.M. Abou-Donia, A.J. Daniels, S.P. Wilson, C. A. Nichol, O.H. Viveros

777

Regulation of Adrenocortical Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin in Normal and Spontaneously Hypertensive Rats M.M. Abou-Donia, A.J. Daniels, C.A. Nichol, O.H. Viveros . 783

Inhibition of Brain Sepiapterin Reductase by a Catecholamine and an Indoleamine S. Katoh, T. Sueoka, S. Yamada

789

XXIX Mouse Mastocytoma Tryptophan Hydroxylase: Role of Iron a n d C a t a l a s e in the E x p r e s s i o n of E n z y m e A c t i v i t y a f t e r A c t i v a t i o n by A n a e r o b i c P r e i n c u b a t i o n w i t h D i t h i o t h r e i t o l H. H a s e g a w a , M . Y a n a g i s a w a , F. I n o u e , A. I c h i y a m a

795

F o l i c A c i d I n d u c e d K i n d l i n g in R a t s : C h a n g e s in B r a i n Amino Acids R . A . O ' D o n n e l l , M . J . L e a c h , A . A . M i l l e r , R.A. W e b s t e r

Impaired Pteroylpolyglutamate Cobalamin-Inactivated Rat

.... 801

S y n t h e s i s in the

J. P e r r y , I. C h a n a r i n , R. D e a c o n , M. L u m b

A Radioimmunoassay Neopterin

for D e t e r m i n a t i o n of

807

D-Erythro-

H. R o k o s , K. R o k o s

Radioimmunoassay

815

for N e o p t e r i n in B o d y F l u i d a n d T i s s u e s

T. N a g a t s u , M . S a w a d a , T. Y a m a g u c h i , T. S u g i m o t o , S. M a t s u u r a , M . A k i n o , N. N a k a z a w a , H. O g a w a

821

A S p e c i f i c A n t i s e r u m A g a i n s t T e t r a h y d r o b i o p t e r i n : Its P r e p a r a t i o n a n d A p p l i c a t i o n to I m m u n o h i s t o c h e m i s t r y S. M a t s u u r a , T. S u g i m o t o , T. N a g a t s u

I. N a g a t s u , T.

Yamaguchi,

827

S p e c u l a t i o n o n the M e c h a n i s m of T h e r a p e u t i c A c t i o n of T e t r a h y d r o b i o p t e r i n in H u m a n D i s e a s e R.A. L e v i n e , W. L o v e n b e r g , H . - C h . A. Niederwieser

Curtius,

833

C o r r e l a t i o n of T e t r a h y d r o b i o p t e r i n C o n t e n t a n d G u a n o s i n e T r i p h o s p h a t e C y c l o h y d r o l a s e A c t i v i t y in C e l l s a n d T i s s u e s D.S. Duch, C.A. Nichol

839

XXX Unconjugated Pteridines in Human Physiological Fluids. Normal and Pathological Profiles J.-L. Dhondt, Z. Bellahsene, C. Largilliere, J. Bonneterre, P. Vanhille, C. Noel, P. Choteau, J.P. Farriaux

845

Biopterin and Phenylalanine Metabolism During Early Liver Regeneration J.-L. Dhondt, G. Kapatos, M. Parniak, H. Wilgus, S. Kaufman

851

GTP-Cyclohydrolase Activities in Rat Liver J.-L. Dhondt, Z. Bellahsene, J.P. Farriaux, M. Deutrevaux

857

Phenylalanine Hydroxylase Assay Using Biopterin and Synthetic Pteridine H.K. Berry, M. Hsieh

863

Dihydropteridine Reductase Levels in Human Normal and Neoplastic Tissues C. Eggar, P.A. Barford, J.A. Blair, A.E. Pheasant, A.E. Guest, D.G. Oates

869

Investigations of Tissue Folates in Normal and Malignant Tissues A.E. Pheasant, J. Bates, J.A. Blair, R. Nayyir-Mazhir .... 875

Blood Cell Biopterin as an Indicator of TransplanationInduced Hemopoiesis and Leukemic Cell Proliferation M. Fink, I. Ziegler, H.J. Kolb, U. Bodenberger, W. Wilmanns

879

XXXI A s s e s s m e n t of U r i n a r y N e o p t e r i n i n t h e E a r l y of H u m a n A l l o g r a f t R e j e c t i o n D. F u c h s , A. H a u s e n , G. R e i b n e g g e r , M. Margreiter, M. Spielberger

Diagnosis

H. W ä c h t e r , C.

Huber, 885

Behaviour of N e o p t e r i n L e v e l s in Patients w i t h Haematological and Gynaecological Neoplasms G. R e i b n e g g e r , D. F u c h s , A. H a u s e n , H. W ä c h t e r , A . B i c h l e r , H. H e t z e l , K. G r ü n e w a l d , C . H u b e r

P t e r i d i n e s in D i c t y o s t e l i u m d i s c o i d e u m a n d polycephalum

891

Physarum

P . L o i d l , D . F u c h s , P. G r ö b n e r , A . H a u s e n , G . H. W ä c h t e r , M. S c h w e i g e r , G. G e r i s c h

Reibnegger,

897

Biochemical and Chemotherapy Studies on BW 301U, a Novel Lipophilic 2,4-Diaminopyridopyrimidine Antifolate D.S. Duch, C.A. Nichol

903

T o x i c i t y of a S e r i e s of A n t i f o l a t e C o m p o u n d s Methotrexate-Resistant Cell Line

in a

M.R. Hamrell

909

Interaction of M e t h o t r e x a t e Poly(L-Lysine) f o r m e d H e p a t i c C e l l s in C u l t u r e

with

Trans-

J.M. Whiteley, J. Galivan, M. Balinska

A Novel Technique Hepatica Cells

for P r o d u c i n g

915

Folate-Deficient

M. B a l i n s k a , W. S a m s o n o f f , J. G a l i v a n

Methotrexate Metabolism

in Acute Lymphoblastic

V.M. Whitehead, D.S. Rosenblatt

921

Leukemia 927

XXXII A l t e r e d Methotrexate Sensitivity in Human K562 Amino A c i d Transport M u t a n t Cells

Leukemic

K . J . S c a n l o n , R. R i e g e l h a u p t , M . P a l l a i , H . S. K a s s a n , S. W a x m a n

Kistard, 933

Methotrexate and Methotrexate Polyglutamates Erythrocytes After High-Dose Methotrexate A. Schalhorn,

in

H. Sauer, W. W i l m a n n s , G. S t u p p - P o u t o t

M e t h o t r e x a t e P o l y g l u t a m a t e s in H u m a n R e v e r s a l of A n t i f o l a t e C y t o t o x i c i t y

941

Fibroblasts:

D.S. Rosenblatt, V.M. Whitehead

947

Human Cells Resistant to Methotrexate: Cross Resistance a n d C o l l a t e r a l S e n s i t i v i t y to the N o n c l a s s i c a l A n t i folates Trimetrexate, Metoprine, Homofolic Acid and CB3717 H. D i d d e n s , D. N i e t h a m m e r ,

R.C. Jackson

953

H o m o l o g y of T w o F o r m s of D i h y d r o f o l a t e R e d u c t a s e i n Human Leukaemic and L1210 Cells During Expression of R e s i s t a n c e to M e t h o t r e x a t e P. L a m e y , N . H a r d i n g , J . D o w

9 59

A d a p t a t i o n of F o l y l p o l y g l u t a m a t e s in Mouse Hepatoma Cells

to F o l a t e

Deprivation

D.G. Priest, M.T. Doig, M. Mangum

965

U t i l i z a t i o n a n d L o s s of R e d u c e d F o l a t e s b y F i b r o b l a s t s in V i t r o

Human

J.H. Hilton, B.A. Cooper, D.S. Rosenblatt,

H. L u e - S h i n g

Factors Affecting Folate Polyglutamate Biosynthesis Rat Liver S. K e a t i n g , D . G . W e i r , J . M .

Scott

.. 971

in 977

XXXIII

Differential Binding of Folates but not Methotrexate (MTX) by Brush Border (BB) and Basal Lateral (BL) Membranes of Rat Renal Cortex R. Corrocher, R.G. Abramson, F.V. King, C. Schreiber, S. Waxman

983

Biosynthesis of Folate Derivatives in Ehrlich Ascites Carcinoma Cells Grown in Mice and in some Organs of the Host E. Sikora, B. Grzelakowska-Sztabert

989

Variability of Thymidylate Synthetase in Ehrlich Carcinoma Cells and its Role in Resistance to 5-Fluorodeoxyuridine M. Jastreboff, B. Kedzierska, W. Rode

995

One-Carbon Metabolism in Human Lymphocytes P.B. Rowe, E. McCairns, D. Sauer, G. Craig

1001

Possible Mechanisms of Folate Catabolism A.E. Pheasant, J.A. Blair, A.M. Saleh, A.E. Guest, P.A. Barford, R. Choolun, R.N. Allan

1007

The Intracellular Folate Pool: Studies of Kinetics and Functional Significance S. Steinberg, S. Fonda, C.L. Campbell, R.S. Hillman

1013

Intestinal Folate Transport. A pH-Dependent, CarrierMediated Process W.B. Strum, H.M. Said

1019

Effects of Folate Depletion on Growth and Survival of Human Cell Line K-562 in Culture D. Watkins, T.E. Shapiro, B.A. Cooper

1025

XXXIV

Alterations in Folate Metabolism of the Rat Induced by Acute Ethanol Administration K.E. McMartin, T.D. Collins

1031

Participation of Pterins in the Control of Lymphocyte Transformation and Lymphoblast Proliferation I. Ziegler, U. Hamm, J. Berndt

1037

The Pterin Component of the Molybdenum Cofactor (Molybdopterin): Relationship to Urothione J.L. Johnson, B.E. Hainline, K.V. Rajogopalan

1043

Report on Presented Papers C.J. Suckling, J.M. Scott

1049

Author Index

1057

Subject Index

1063

TRANSPORT OF FOLATE AND PTERIN COMPOUNDS

F.M. Huennekens, M.R. Suresh*, C.E. Grimshaw, D.W. Jacobsen, E.T. Quadros, K.S. Vitols and G.B. Henderson Division of Biochemistry, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, CA 92037

I.

Introduction

This

contribution

to

the

Seventh

International

Pteridine

Symposium honors a most remarkable man, Sir Frederick Gowland Hopkins.

His

formidable

achievements

in

research

were

appropriately acknowledged via the Nobel Prize in 1929, but he performed guiding

perhaps the

an

even

activities

Biochemistry

at

Biochemistry

(1),

Cambridge a

greater

of

an

service

to

science

by

Department

of

Perspectives

in

outstanding

University.

collection

of

essays

from

Hopkins'

colleagues, provides an eloquent testimony to his ability to select young scientists with potential. Hopkins' career was his

interest

A less known facet of

in pteridines.

An

ardent

student of Nature, he was fascinated by the brilliant pigments in butterfly wings

and,

although lacking

the advantages

of

current technology, he succeeded in isolating and partially characterizing two of these compounds, xanthopterin and leucopterin

(2).

He may be regarded, therefore,

as one of

the

founders of the pteridine field. A considerable body of information about pteridines and the related

folate

observations.

compounds Much

is

has accumulated now

known

since

about

the

those

early

chemistry,

£ Present address: Department Alberta, Edmonton, Canada.

of

Chemistry and Biology of Pteridines © 1983 Walter de Gruyter & Co., Berlin • New York

Pharmacy,

University

of

2

Sir Frederick Gowland Hopkins (1937) enzymology, transport, biological function and clinical use of these interesting Pteridine

materials.

Symposium will

Our contribution to the present

focus upon the transport

systems,

primarily in L1210 mouse leukemia cells and in Lactobacillus casei.

Experimental

observations,

summarized briefly, will

provide a basis for discussion of possible mechanisms and suggestions for future work.

II. Transport of Folate Compounds A.

Relationship to intracellular metabolism and Methotrexate

cytotoxicity. Mammalian

cells

require

a

source

of

tetrahydrofolate,

coenzyme form of the vitamin, for replication.

the

Under physio-

logical conditions, cells are nurtured by folate or 5-methyltetrahydrofolate

(Fig. 1), but

in the laboratory,

5-formyl-

tetrahydrofolate (folinate) can also be used to support cell growth.

Pathways for the conversion of these precursors to

tetrahydrofolate are outlined in Fig. 2. tetrahydrofolate

via

the

Folate is reduced to

NADPH-dependent

dihydrofolate

reductase (EC 1.5.1.3); this enzyme assumes special importance

3

H,N

.N.

JIL

Tf' l ^UA

5

C00H i ' / v 0 iLcH2-NHf\c-NH-fH N

X

H

W

Folate

(¿H2)2 COOH

N^J^N^-CH2 —N NH2

Methotrexate

CH3

v H « 11 I H ""•^N^"

5-Formy1 tetrahydrofol ate (Folinate)

CHO

N " Y

> LH

H

$-Methyltetrahydrofolate

N CH3

Fig. 1.

Structures of folate

compounds.

as the target for the cancer chemotherapeutic agent, Methotrexft it

ate (MTX)

.

5-Methyltetrahydrofolate also requires the inter-

vention of only a single synthetase contrast, direct,

(EC the

pathway

requiring

methenyl

enzyme,

2.1.1.13),

derivative

tetrahydrofolate

to

from

the B ^ - d e p e n d e n t

generate

5-formyltetrahydrofolate

conversion (catalyzed

of

the

by

substrate

to

the ATP-dependent

Of the three enzymes cited above, only dihydrofolate been

bacterial sively

purified

is

the

less 5,10-

methenyl-

to

homogeneity

from

both

level. reductase

mammalian

sources, thus allowing it to be characterized

(3).

Ir

synthetase (EC 6.3.3.2)), followed by loss of

the C-^ group at the methenyl or methylene oxidation

has

methionine

tetrahydrofolate.

Methionine

synthetase

has

been

obtained

and

extenfrom

AA Abbreviations: MTX, Methotrexate; pABG, £-aminobenzoylglutamate; PBS, phosphate-buffered saline; HEPES, N-2-hydroxyethylpiperazine-N'-ethanesulfonate; pCMS, £-chloromercuriphenylsulfonate; Me2S0, dimethylsulfoxide.

4 F

Fig. 2. Pathways for production of tetrahydrofolate. Abbreviations: F, folate; FH^, tetrahydrofolate; CH3-FH11, 5-methyltetrahydrofolate; CHO-FHi,, 5-formyltetrahydrofolate; CH-FHI+, 5 ,10 -methenyltetrahydrofolate; MTX, Methotrexate; B12s,cob(I)alamin. Other abbreviations are standard. Escherichia

coli

levels

the

of

K-12 enzyme

eukaryotic cells. (and its growing

in highly have

purified

hampered

form

its

(4), but

isolation

low from

However, the obligatory role of this enzyme

cofactor) in the replication of mammalian cells on

5-methyltetrahydrofolate

has

been

established

recently using cultured L1210 mouse leukemia cells as a model system

(5).

Methenyltetrahydrofolate

synthetase

partially purified some years ago from Micrococcus (6)

and

from

properties

sheep

liver

(7),

but

have remained obscure.

a

number

aerogenes

of

critical

The use of folinate as a

"rescue" agent in high-dose MTX therapy has revived in this enzyme,

and recently

our

on

enzyme

immobilized

is

homogeneous

folinate upon

interest

laboratory has purified

8000-fold from L. casei using sequential raphy

was

and

ATP

affinity

chromatog-

(unpublished).

SDS-polyacrylamide

it

gel

The

electro-

phoresis and has a molecular weight of 23,000 under these conditions; there

is no

evidence

enzyme

number

is 100 min"^,' and the K m values for folinate

are

0.8

and

1.0

are

MM,

able

to gel

aggregates when

native

phosphates

is subjected

of larger

filtration.

respectively; to

replace

ATP

other with

The

turnover and ATP

nucleoside varying

the

tri-

degrees

5 of efficiency. Current studies are directed toward elucidation of the mechanism of the novel reaction (equation 1) catalyzed

N I I HC=0

N

+

ATP

4-

,

M2+

\/ N

H

C H

by this enzyme.

V N /

+

ADP

3-

+

P.

2-

(1)

Most ATP-dependent reactions do not involve

chromophoric substrates, but the distinctive spectra of both the reactant ( ^

at 286 nm) and the product ( ^

at 359 nm)

in the above reaction suggests that the putative intermediate(s) might be detectable by stopped-flow spectrophotometry. Studies iji vitro on the cytotoxicity of MTX involve exposure of cells to the drug and one of the substrates shown in Fig. 2 (usually folate itself). same

system

for

entry

MTX and folate compounds share the into

the

cells

(see

II-B),

and

the

transport process is an important factor in determining drug effectiveness

(8).

Since

the transport

system

has

a

high

affinity for reduced folate compounds and a low affinity for folate,

the

drug

folate-grown cells. conditions interdict folate

is

should

be

more

effective

against

The advantage of MTX under the

strengthened

by

the

ability

of

the

latter

drug

to

the metabolic conversion of folate to tetrahydro-

(cf. Fig. 2).

experimentally.

These

considerations

are

borne

out

In L1210 cells grown on folate the ID^^ for

MTX is 13 nM, which may be compared to corresponding values of 19

and

24

nM

for

growth

5-formyltetrahydrofolate. substrate

was

used

on

5-methyltetrahydrofolate

and

In these experiments, each folate

at an optimal

level

for propagation

of

L1210 cells (5). B.

General characteristics.

Transport

of

folate

compounds

has

been

studied

in

eukaryotic and prokaryotic systems (reviewed in 8-10).

various Plots

6 of initial rate (v) vs. substrate concentration saturation kinetics. concentration transport",

(S) exhibit

This observation and the achievement of

gradients i.e.,

define

the

energy-dependent

substrate-binding protein.

process and

as

"active

mediated

by

a

The v vs. S data yield V_„ v and K^. —

—

JTLAX

T

values (the latter corresponding to the substrate concentrations at which the rates of transport are half-maximal) that characterize

the

respective

systems.

Table

I

summarizes

representative K^ values for several mammalian cells (LI210, hepatocytes and enterocytes) and a bacterium (L. casei).

It

should be noted that K^ values for L1210 cells are markedly affected by the buffer in which the measurements are made (see IV).

In general, folate compounds are approximately equiva-

lent as substrates for the bacterial system, whereas 5-methyltetrahydrofolate by

the

and

mammalian TABLE I.

5-formyltetrahydrofolate

cells;

MTX,

for

reasons

are

not

preferred

yet

clear,

Kt VALUES FOR TRANSPORT OF FOLATE COMPOUNDS BY VARIOUS CELLS Kt

Cell L. casei

CH3-FH4

CHO-FH 4

Folate

MTX

pM

PM

pM

pM

0.0 6*

0 .09

0.05

11

LI 210

1.5

5

100*

5

8,9,12

L1210**

0.3

0.5

14

0.7

13

240*

10 and 2000

14-17

Rat hepatocyte Rat hepatoma H35 Mouse enterocyte Rat enterocyte (vesicles)

0.2

*

Ref.

5 2.5 - -

1.4*

- -

- -

10.4*

18

- -

- -

87

19

1.5

20

0.4

Abbreviations as defined in legend for Fig. 2. is

Measured as K^, using labeled MTX or folate as substrate.

**Measured in Mg-HEPES-sucrose buffer: 20 mM HEPES (adjusted to pH 7.4 with MgO) plus 225 mM sucrose. --Not determined.

7

behaves

like

systems.

a

reduced

folate

compound

in

the

eukaryotic

Low affinity systems (see data for hepatocytes

Table I), characterized by high V anion transporters.

IuaX

in

values, may be general

Mammalian and bacterial cells display a broad specificity with respect

to

the

uptake

of

folate

instance a single system appears major

members

of

this

group

compounds,

and

to accommodate

(folate,

in

each

all of

the

5-methyltetrahydro-

folate, 5-formyltetrahydrofolate and MTX).

This

generaliza-

tion corrects earlier statements ,that folate and the reduced folates were transported by separate systems in L1210 (8-10).

The previous belief

consistent observations:

stemmed

from

several

cells

mutually

(A) The reported K t and V m a x

values

for folate, although showing wide variance, were considerably higher

than those for the reduced folates; (B) MTX and the

reduced folates were relatively ineffective as inhibitors of folate uptake and vice versa. (C) Uptake of reduced folates, but not folate, was sensitive to mercurials; and (D) Mutants selected for resistance to MTX (due to impaired transport of the drug) showed good uptake of folate.

Recent work from our

laboratory (21) has resolved this problem. rations

of

[ H]folate

are

Commercial prepa-

contaminated

with

breakdown

products, identified by TLC and HPLC as 6-substituted pterins 9 10 and pABG. Degradation, which involves cleavage of the C -N bond, appears to result from radiolysis, since it occurs even when the purified material is stored frozen, is more severe in samples of higher specific activity, and can be suppressed by ascorbate.

Upon removal of impurities, the true picture of

folate uptake emerges (Fig. 3).

The K t value for folate is 14

yM in Mg-HEPES-sucrose buffer (Table I), 140 uM in HEPES-KC1, and 400 yM in PBS.

Its uptake is inhibited competitively by

MTX and non-competitively by pCMS. Small amounts of impurities ( < 51) ordinarily do not produce such

large

perturbations

in

substrate

uptake.

In

this

8 instance, kinetic

however,

there

parameters.

is

One

6-hydroxymethylpterin,

is

an

unfortunate

of

the

taken

combination

principal

up

by

an

of

impurities,

active

transport

system, separate from that responsible for folates, which has a

Vmax

of

higher

200

than

pmoles/min/mg

the

V_a,, indx 6-hydroxymethylpterin,

for

protein;

folate

moreover,

lower than the K t for folate. in

[ H]MTX,

but

these

this

is

transport. is

20

yM,

ca. 20-fold The

Kt for t value 7-fold

a

Similar pterin impurities occur

are less troublesome

since K t

for

the

drug is ca. 5 yM.

III. Transport of Pterins Pterin

transport

recently

with

(22).

pterin

K^

various

L1210

respect

specificity have

by

other •7

to

cells

kinetic

Labeled

values

pterins

of have

has

been

characterized

parameters

and

substrate

6-hydroxymethyl-

and

6-formyl-

20

and

been

52

pM,

tested

uptake of [ H]6-hydroxymethylpterin

respectively,

as inhibitors of

(Table

II).

This

and the

system

appears to have the highest affinity for pterins bearing small substituents exhibiting

at

C-6,

substrate

into the cells.

and

it

is

competition

possible are

that

themselves

compounds transported

Adenine is a potent inhibitor (K^ = 20 uM) of

6-hydroxymethylpterin uptake, which is perhaps not in view true,

of

the

structural

similarity.

however,

indicating

that

pterins

adenine transport system in L1210 cells.

The do

surprising

converse not

is

not

utilize

the

Future studies will

be directed toward isolation of the putative pterin

transport

protein and toward elucidation of the metabolic role of the 6substituted

pterins.

IV. Anion Effects in the Transport of Folate Compounds Transport

of

folate

compounds

in

L1210

cells

is

influenced

9

5

10

15

20

Time, min

Fig. 3. Transport of commercial and purified folate in L1210 cells. Assay mixtures contained cells (1.5 x 10 7 ) and 100 nmoles of [3',5',7,9- 3 H]folate (obtained commercially or purified as described below) in 1.0 ml of 20 mM HEPES-140 mM KC1, pH 7.4. In each case, folate samples were adjusted to a specific activity of 30,000 cpm/nmole. Assay mixtures were incubated at 37° for the indicated times, and transport was terminated by the addition of 9 ml of ice-cold 140 mM KC1. Cells were recovered by centrifugation at 1000 x £ (5 min, 4°) and analyzed for radioactivity. Purification of labeled folate was achieved by chromatography on Eastman cellulose sheets (20 x 20 cm) using 50 mM HEPES, adjusted with KOH to pH 7.4, as the solvent system. From (23). markedly by anions (9,24).

This is shown in Fig. 4, in which

3

the uptake of [ H]MTX is compared in different buffers.

PBS

was quite inferior to HEPES, as judged by both the rate and extent of uptake of the substrate.

Since PBS contains several

anions, it was suspected that the latter might be acting as inhibitors.

Using HEPES as a standard, various anions were

then tested and found to be inhibitory; the middle curve in Fig. 4 illustrates the deleterious effect of a representative anion, CI".

Double reciprocal plots of these data indicated

that anions were competitive inhibitors, affecting K t values but not V m a x of the transport process; some representative

K^

values for anions acting as inhibitors of C^hImTX uptake are given in Table III.

10

TABLE II.

INHIBITION OF 6-HYDROXYMETHYLPTERIN TRANSPORT BY OTHER PTERINS

Compound Pterin 6-Hydroxypterin (Xanthopterin) 6-Formylpterin 6-Methylpterin 6-Carboxypterin Biopterin Tetrahydrobiopterin 6,7-Dimethyltetrahydrobiopterin

K.l UM 30 52 42 100 350 180 1100 680

Transport of [3H]6-hydroxymethylpterin was measured in assay mixtures containing cells (1.6 x 10 7 ), 10 ¿¿M [3H] 6-hydroxymethylpterin (specific activity 65,000 cpm/nmole), and other components as indicated. Suspensions were incubated at 37° for 2 min, and transport was terminated by the addition of 9 ml of ice-cold 0.15 M KC1. Supernatants obtained by centrifugation were discarded, and radioactivity associated with the cell pellets was measured. K. values were determined from Dixon plots at 2 different substrate concentrations (5 and 10 nM) and in the presence of varying amounts of the indicated substrate analogs. Mercaptoethanol (10 mM) was also present when reduced biopterins were tested. From (22).

Fig. 4. Time-dependent uptake of MTX by L1210 cells in various buffers. Uptake measurements were performed as described in the legend for Table III. Buffer compositions: HEPES, 160 mM HEPES adjusted to pH 7.4 with KOH; HEPES-KC1, 20 mM HEPES and 140 mM KC1 adjusted to pH 7.4 with KOH; PBS, 138 mM NaCl, 2.7 mM KC1, 8.1 mM Na2HP0it, 1.5 mM K^POit, 1.0 mM C a C l 2 , and 0.5 mM MgCl 2 . From (21).

11

sitivity. anions).

(The L. casei

is unresponsive

to

The use of HEPES buffers allows concentration ratios

approaching

100

strengthening ratios)

system, however,

to

be

earlier

that

this

achieved

in

evidence

system

L1210

(based

mediates

cells,

thereby

much

smaller

upon

active

transport.

The

possible objection that HEPES is not a "physiological" buffer has been met by recent observations in this medium

remain

viable, neither

maintain the ability to metabolize

Although a wide variety folate compounds,

(unpublished) that swell

nor

cells

shrink,

of anions can

inhibit

the

influx

some are also able to affect their and AMP serve as

of

efflux,

but in the latter instance the action is stimulatory (25). Fig. 5, 5-formyltetrahydrofolate

and

glucose.

In

represent-

ative agents to illustrate the enhancement of efflux of preloaded

C 3 H] MTX.

surprising,

but

Results the

equal

with

the

former

effectiveness

unrelated compound, AMP, was quite novel. gation

using

several

other

anions with

compound of

a

not

structurally

A further

this

were

investi-

capability

(see

Table III) revealed that the concentrations required for halfmaximal stimulation of efflux ( K s t i m ) were very similar to the concentrations

for

half-maximal

inhibition

of

influx

(K.).

Fig. 5. Time-dependence for the stimulation of MTX efflux by AMP and 5-formyltetrahydrofolate. Cells were preloaded with MTX by incubation with 5.0 nM [ 3 HJMTX for 20 min at 37° in a HEPES buffer (160 mM HEPES and 2 mM M g C l 2 , adjusted to pH 7.4 with KOH) containing 0.5 mM glucose. From (25).

12

TABLE III. KINETIC CONSTANTS DERIVED FOR EFFECTS OF VARIOUS ANIONS ON MTX INFLUX AND EFFLUX K i for Anion

MTX

K„ + • for st lm MTX

Influx MTX Folate 5-Formyltetrahydrofolate AMP ADP Thiamine pyrophosphate Phosphate Sulfate Chloride

Maximum Stimulation of Efflux

fiM

Efflux MM

0.6 20 1.2

0.8 30 2.5

2.7 2.0 3.2

40 34 5

125 100 8

3.7 2.0 4.0

600 550 32000

1600 2000 40000

3.2 2.8 3.6

-fold

MTX influx was determined in assay mixtures (1.0 ml, final vol) that contained cells (2 x 10 7 J, 2 (iM [ 3 HjMTX, and variable amounts of the indicated anion in HEPES-sucrose buffer (20 mM HEPES, 225 mM sucrose, and 2 mM MgCl2, adjusted to pH 7.4 with KOH). After incubation for 5 min at 37°, the cells Were diluted with 8 ml of ice-cold 0.15 M KC1, collected by centrifugation, and analyzed for radioactivity. K j values were calculated from Dixon plots of the reciprocal of the transport rate vs. inhibitor concentration. For efflux determinations, cells" were preloaded by incubation for 15 min at 37° in 160 mM HEPES, pH 7.4, containing 2 mM MgCl2, 0.5 mM glucose, and 5 jxM [ 3 H]MTX, washed, and suspended at 37° in HEPES-sucrose buffer. Time-dependent release of accumulated [ 3 HjMTX was then measured by transfer of aliquots into 5 ml of 0.15 M KC1 at the indicated time intervals, collection of the cells by centrifugation and analysis for radioactivity. Efflux parameters were calculated from a double-reciprocal plot of the stimulation of efflux _vs. anion concentration as described in (25). Also

evident

from

these

data

is

the

fact

that

all

of

the

agents produce approximately the same degree of stimulation, viz., 2- to 4-fold.

Examples

of good

inhibitors of

influx

which do not stimulate efflux include ATP, citrate, NAD, and fructose diphosphate.

The relevance of these anion effects to

the mechanism of transport is discussed subsequently (see VI).

13

V.

Binding proteins from L. casei and L1210 cells.

Previous work with the folate transport

system

in L. casei

culminated in the isolation of a membrane-associated folatebinding protein (26).

This extremely hydrophobic protein was

extracted

cells

Triton

from

X-100

whole

and

purified

or to

membrane

adsorption and elution from microgranular step.

preparations

homogeneity,

making

with

use

of

silica as the key

The protein could be readily detected and quantitated

in cells by binding measurements using this procedure

could not be used

to

[^H]folate (27), but follow

encapsulated protein during purification.

the

detergent-

Admixing the cells

with [^Hlfolate prior to extraction, however, led to retention of the label by the protein in the detergent micelles and, at the final stage in the purification, the protein contained 1 mole of noncovalently-bound M f o l a t e per mole of protein (MW 25,000).

The amino acid composition of this protein has been

determined, and if it can be sequenced and crystallized, the three-dimensional structure should become available. Success in the above project was facilitated by the relatively large amount of binding protein in the cells (120

pmoles/mg

protein) and its high affinity for folate (K^ = 1 nM). favorable logistics that exist for the counterpart

Less

in L1210

cells (1 pmole/mg protein; K^ = 100 nM for 5-methyltetrahydrofolate)

greatly

diminished

the

likelihood

that

the

procedure could be employed for its purification.

above

According-

ly, methods have been developed for marking the L1210 binding protein with a covalently pounds,

activated

carbodiimide

with

in Me 2 S0,

purpose (28).

attached substrate.

were

Folate

com-

l-ethyl-3(3-dimethylaminopropyl) found

to be

suitable

for

this

Irreversible inhibition of the transport system

by exposure of the cells to activated MTX is shown in Fig. 6. The effect is both time- and concentration-dependent. parison shows

of

that

several their

carbodiimide-activated effectiveness

as

A com-

folate

substrates

irreversible

inhibitors

14

Fig. 6. Time-dependent loss of MTX transport activity in cells pretreated with activated MTX. Activated MTX was prepared by combining MTX and l-ethyl-3(3-dimethylaminopropyl) carbodiimide (4 and 40 mM final concentrations, respectively) in 0.5 ml of Me2SO and incubating the solution for 30 min at 23°. Aliquots (0.01 ml) of the reagent at the indicated concentrations were then added to cells (2 x 10 7 ) suspended in 0.99 ml of 160 mM HEPES buffer, pH 7.4. After incubation at 37° for the indicated times, the cells were recovered by centrifugation, washed with HEPES buffer, and analyzed for MTX transport. Transport of MTX is plotted on a log scale as a function of time. From (28).

Fig. 7. Inhibition of MTX transport by activated aminopterin, MTX, folate, and pABG. Generation of activated compounds, treatment of cells with the indicated concentrations of these compounds, and MTX transport determinations were performed as described in the legend for Fig. 6. From (28).

15

follows

the- same

relative

affinity (Fig. 7). has

been

verified

below) and by lack (e.g.,

glucose,

however, this

reagent.

were

added

unlabeled

substrate

of

to

their

reversible

protection

inhibition

of

cells

in

the

amounts

absence

about

301

the

transport

of

presence total

[ ]MTX

of

recently,

reagent,

by

3

excess

radioactivity

MTX.

improved

binder,

activated

into the membrane could be blocked by an

(see

systems

folate

incorporated More

procedure

in the membrane

and

of

binding

experiments

The

labeled

increasing

only

other

and phosphate).

the only protein When

MTX,

as

The specificity of the labeling by

leucine

is not

order

the

unlabeled N-hydroxy-

succinimide ester of MTX, has been shown to maintain the high level of labeling of the transport protein while diminishing this

the amount

reagent

in

of background

conjunction

with

considerably

(unpublished).

[^HjMTX

of

high

Use

of

specific

activity (20 C^/mmole) allows the folate binder to be detected during extraction and purification. cation procedure

without

Repetition of the purifi-

the pre-labeling

step

should

the native protein suitable for physical and chemical

yield

charac-

terization, antibody preparation, measurement of the fidelity of binding constants after reconstitution into liposomes, and testing

of

the hypothesis

that

a

single

protein

transports

folate compounds and various anions (see VI).

Procedures cells

for

are being

studying

folate

explored

binding

in this

proteins

laboratory.

A

in

single

fluorescein

derivative of MTX, originally developed for labeling dihydrofolate reductase (29), has a strong affinity for the transport protein (25), but it is internalized very slowly (30). this

agent

porters

on

may

be

cells

capable

of marking

and

discriminating

of

outward-facing via

Thus, trans-

fluorescence

microscopy between individual cells with respect to transport competence.

Another approach would be to use Sephacryl

beads

(diameter, 70 (im) derivatized with MTX or 5-formyltetrahydrofolate

to

visualize

5-methyltetrahydrofolate,

the

membrane-associated

although

the optimal

binders;

substrate

for

16

the transport system, would be less suitable for this purpose because of its susceptibility bility

of

this

approach

has

to air-oxidation. been

The feasi-

demonstrated

by

previous

experiments in which Sephacryl beads derivatized with vitamin (and coated with

transcobalamin-II)

detect B 1 2 -transcobalamin-II

receptors

have

been

used

(31) on single

to

L1210

cells (Fig. 8).

Fig. 8. Scanning electron micrograph (x700) of L1210 cells bound to Sephacryl-aminopropylcobalamin beads. Experimental details given in (31). VI

Mechanism

Characteristics of the folate transport systems in L. casei and

L1210

cells,

as

described

above,

provide

proposing mechanisms for these processes. the membrane-associated

binding

internalization

substrate

of

the

protein (with

a basis

for

In the bacterium is responsible cation

for

cotransport

(32)), and the system appears to be regulated by repression and derepression of synthesis of this protein.

An accessory

factor is involved in coupling the energy source (ATP) to the transport protein (33) ; the nature of this linkage, however, is not yet clear.

L1210 cells also utilize a membrane binder,

but regulation appears to be mediated by cAMP (34), and the

17 Other

Fig. 9.

driving

force

is an

~P

Energy transduction.

anion

gradient.

However,

the

facile

transduction of energy between ATP and ion gradients (Fig. 9), as exemplified by the ability gradients, or H

gradients

phosphorylation),

makes

of ATPases

to produce ATP

it

difficult

to

to create

Na /K

(as in oxidative assign

a

precise

energy source to each of these transport processes. In L1210 cells substrate translocation is thought to occur by an anion

exchange mechanism

(25,35).

In this

hypothesis,

influx of a folate molecule is paired with efflux of an intracellular anion (e.g., P^, HCOj" or CI"); this process

(like

cation co-transport in L. casei) also serves to maintain the membrane potential.

Although technical problems have made it

difficult to demonstrate the required 1:1 stoichiometry, the following

experimental

exchange mechanism:

observations

support

the

anion

(A) Both the binding and transport

of

folate compounds are inhibited competitively by a variety of anions; (B) Some (but not all) of these anions also stimulate the efflux of previously the

values

stimulation

of

internalized folate compounds,

(concentrations

the

rate

of

required

efflux)

of

for these

and

half-maximal anions

are

approximately equal to the K^ values for inhibition of influx. Moreover, the stimulatory ability of these anions is lost when cells are pre-treated with activated folate substrates.

(C)

Minor

are

components

of

phosphate

and

sulfate

transport

18

inhibited competitively by MTX (with a K^ value nearly equal to

the

Kt

for

MTX

transport),

and

these

components

are

inhibited irreversibly by activated MTX. The

simplest

explanation

of

these

data

is

that

transport

proceeds via an exchange process and that it is mediated by a single protein with a high affinity for folate compounds and lower affinities for other anions.

A similar mechanism has

been postulated for the exchange of HCO^" and Cl" in erythrocytes

(36).

However,

the lack of precedent for such broad

specificity at a binding site, and the fact that only a few anions

(of

the

many

tested

(35))

are

actually

capable

of

stimulating substrate efflux, suggests that modifications of this hypothesis may be necessary. protein while

might

those

actually few

anions

For example, the binding

translocate capable

of

only

folate

being

compounds,

coupled

to

this

process would utilize one or more separate transport proteins. This modification would require, however, a binding site on the folate protein with sufficient flexibility to permit competitive inhibition by a variety of anions, and some mechanism for linking the operation of the folate and anion binders. There

is

systems.

ample

precedence

for

multi-component

transport

Hexose transport in E. coli involves a soluble cyto-

plasmic protein (HPr), whose phosphorylated form is coupled, via a membrane component, to other membrane proteins specific for the various hexoses (37).

In L. casei, moreover, a second

membrane

with

component

interacts

the

binding

protein

for

folate, as well as with those for thiamine and biotin (33). Even when the above questions are resolved, there will remain the more difficult task of delineating the detailed mechanism of folate transport, i.e., the spatial and temporal

sequence

of events by which the substrate

is translocated across the

membrane.

In

is

transport

and

this

context,

enzyme

it

catalysis

instructive

(Fig. 10).

to

compare

Each

process

involves the binding of a low molecular weight substrate by a

19

specific thereof),

protein,

movement

and release

of

of

the

the

substrate

substrate

(or

(or

a

portion

product).

The

protein must then return to its original position (or state) in order to accommodate the next substrate molecule.

Enzyme catalysis

E

+ S

Transport

TP +

—

ES

EP

—

E

. T p- Sl

—

TP + S i

-

S Q — - TP.So

+ P

Fig. 10. Comparison of transport and enzyme catalysis. Abbreviations: E, enzyme; S, substrate; P, product; TP, transport protein; and S and S-, substrate outside and inside, respectively. The

membrane-associated

binding

protein

fulfills

functions during the various stages of substrate tion:

several

transloca-

(A) Facing outward, the protein binds and envelops the

folate compound to shield charged or hydrophilic groups from the lipid interior of the membrane.

The binding site utilizes

a combination of functional groups, perhaps similar to those in folate-dependent enzymes.

(B) The protein undergoes a con-

formational change in which the binding site, containing the shielded

substrate,

is

turned

inward.

(C) Release

of

the

substrate could occur spontaneously or it may be assisted by some intracellular factor.

(D) The transporter may return to

the outward-facing configuration by binding and effluxing an anion, or it may return empty (as appears to be the situation in L. casei). The sequence in Fig. 10 is also relevant to the time-frame and energetics of the transport system.

Calculations based upon

the rate of uptake and the number of transporters per cell indicate

that

in L.

molecule

requires

casei

a number

and

L1210

of

seconds

What is the rate-determining step?

cells to be

each

substrate

internalized.

One might guess that it is

a conformational change in the protein rather than substrate

20 binding or release (which, by analogy with enzyme catalysis, should be in the sub-second range). with respect to the energetics. profile

is higher

than

the

The same questions arise

The final state in the energy

initial

state,

since

substrate

translocation is concentrative or "uphill", but it is not yet clear which are the energy-requiring and energy-yielding steps in the transport sequence. Acknowledgments The authors are indebted to E. Zevely, G. Hansen, and Y. Montejano for expert technical assistance. Experimental work described in this paper was supported by grants from the National Institutes of Health (CA06522, CA16600, CA23970, CA32261, AM25406) and the American Cancer Society (CH-31 and CH-229). M.R.S. was the recipient of a Junior Fellowship from the California Division, American Cancer Society. References 1.

Needham, J., Green, D.E., eds.: Perspectives in Biochemistry, University Press, Cambridge, 1937.

2.

Schopf, C.: in Pteridine Chemistry (W. Pfleiderer and E.C. Taylor, eds.) pp. 3-14, MacMillan, New York, 1964.

3.

Gready, J.: (1980).

4.

Fujii, K., Huennekens, F.M.: 6745-6753 (1974).

5.

Fujii, K., Nagasaki, T., Huennekens, F.M.: Chem. 256, 10329-10334 (1981).

6.

Kay, L.D., Osborn, M.J., Hatefi, Y., Huennekens, F.M.: J. Biol. Chem. 235, 195-201 (1960).

7.

Greenberg, D.M., Wynston, L.K., Nagabhushanam, A. Biochemistry 4, 1872-1878 (1965).

8.

Huennekens, F.M., Vitols, K.S., and Henderson, G.B.: Adv. Enzymol. 47, 313-346 (1978).

9.

Goldman, I.D.: (1971).

10.

Sirotnak, F.M., Chello, P.L., Brockman, R.W.: Cancer Res. 16, 381-447 (1979).

11.

Henderson, G.B., Zevely, E.M., Kadner, R.J., Huennekens, F.M.: J. Supramol. Struct. 6, 239-247 (1977).

12.

Sirotnak, F.M., Donsbach, R.C.: (1976).

Adv. Pharmacol. Chemother. 17, 37-102 J. Biol. Chem. 249, J. Biol.

Ann. N.Y. Acad. Sci. 186, 400-422 Methods

Cancer Res. 36, 1151-1158

21

13.

Henderson, G.B., Zevely, E.M.: in press.

Arch. Biochem. Biophys.,

14.

Quadros, E., Huennekens, F.M.:

Unpublished results.

15.

Gewirtz, D.A., White, J.C., Randolph, J.K., Goldman, I.D.: Cancer Res. 40, 573-578 (1980).

16.

Hörne, D.W., Briggs, W.T., Wagner, C.: J. Biol. Chem. 253, 3529-3535 (1978).

17.

H o m e , D.W., Briggs, W.T. , Wagner, C.: Res. Commun. 68, 70-76 (1976).

18.

Galivan, J.:

19.

Chello, P.L., Sirotnak, F.M., Dorick, D.M., Donsbach, R.C.: Cancer Res. 37, 4297-4303 (1977).

20.

Seihub, J., Rosenberg, I.H.: J. Biol. Chem. 256, 4489-4493 (1981).

21.

Huennekens, F.M., Vitols, K.S., Suresh, M.R., Henderson, G.B.: in Molecular Actions and Targets for Cancer Chemotherapeutic Agents (A.C. Sartorelli, ed.) pp. 333-347, Academic Press, New York, 1981.

22.

Suresh, M.R., Huennekens, F.M.: 4, 533-541 (1982).

23.

Huennekens, F.M., Suresh, M.R., Vitols, K.S., Henderson, G.B.: Adv. Enzyme Regul. 20, 389-408 (1982).

24.

Henderson, G.B., Zevely, E.M.: 200, 149-155 (1980).

25.

Henderson, G.B., Zevely, E.M.: Biochem. Biophys. Res. Commun. 99, 163-169 (1981).

26.

Henderson, G.B., Zevely, E.M., Huennekens, F.M.: J. Biol. Chem. 252, 3760-3765 (1977).

27.

Henderson, G.B., Zevely, E.M., Huennekens, F.M.: Biochem. Biophys. Res. Commun. 68, 712-717 (1976).

28.

Henderson, G.B., Zevely, E.M., Huennekens, F.M.: J. Biol. Chem. 255, 4829-4833 (1980).

29.

Gapski, G.R., Whiteley, J.M., Rader, J.I., Cramer, P.L., Henderson, G.B., Neef, V., Huennekens, F.M.: J. Med. Chem. 18, 526-528 (1975).

30.

Henderson, G.B., Russell, A., Whiteley, J.M.: Biochem. Biophys. 2£2, 29-34 (1980).

31.

Jacobsen, D.W., Montejano, Y.D., Vitols, K.S., Huennekens, F.M.: Blood 55, 160-163 (1980).

32.

Henderson, G.B., Potuznik, S.: 1098-1102 (1982).

33.

Henderson, G.B., Zevely, E.M., Huennekens, F.M.: J. Bacteriol. lZl_t 1308-1314 (1979).

Biochem. Biophys.

Cancer Res. 41, 1757-1762 (1981).

Biochemistry International

Arch. Biochem. Biophys.

Arch.

J. Bacteriol. 150,

22 34.

Henderson, G.B., Zevely, E.M. , Huennekens, F.M.: Cancer Res. 38, 359-361 (1978).

35.

Henderson, G.B.: in Proceedings 13th International Cancer Congress, Alan R. Liss, New York, in press.

36.

Passow, H., Fasold, H. , Gartner, E.M., Legrum, B., Ruffing, W., Zaki, L.: Ann. N.Y. Acad. Sci. 341, 361-383 (1980).

37.

Roseman, S.: in Metabolic Pathways, L.E. Hokin, ed., Vol. VI, pp. 41-89, Academic Press, New York, 1972.

SYNTHETIC METHODS IN PTERIDINE CHEMISTRY:

SOME APPLICATIONS

TO PTERIDINE NATURAL PRODUCTS

Edward C. Taylor Department of Chemistry, Princeton University, Princeton, N.J. 08544

In my talk this morning I will present a survey of the principal methods currently available for the construction of the pteridine ring system, along with comments on the limitations and advantages of each procedure.

Except for a few special

cases where construction of a multifunctional substituent at position 6 constitutes the major synthetic challenge for the target compound involved, I will not attempt to cover the manipulation of substituents on the pteridine ring itself. Professor Pfleiderer, in his start-of-the-art lecture, will be discussing this feature of pteridine chemistry.

Since the

major emphasis of this talk will be on synthetic methods suitable for the construction of pteridines of biological interest (primarily pterins), I will omit those synthetic methods which are not general, or which are restricted to types of pteridines (such as 1,3-dimethyllumazines) not widely found in nature. The classical synthetic route to pteridines involves condensation of a 4,5-diaminopyrimidine with an a,6-dicarbonyl compound (Fig. 1).

Its usefulness and flexibility result from

the wide diversity of substitution patterns possible with both components and the very high yields which are generally obtained.

This so-called Isay condensation has been used,

over the history of pteridine chemistry, for the preparation of the majority of known synthetic pteridines.

Chemistry and Biology of Pteridines © 1983 Walter de Gruyter & Co., Berlin • New York

When the

24

Pteridines from Pyrimidines. The Isay Condensation

HN-^VNH2

+

ArCH2Cl

R J_fi Ra

^

ArCH2S02CH2Ar

H N ^ V V R ! (R2)

»-

0