Unconjugated pterins and related biogenic amines: proceedings of the First International Workshop, Flims, Switzerland, february 28 - march 7, 1987 9783111503844, 9783110113419

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Unconjugated Pterins and Related Biogenic Amines

Unconjugated Pterins and Related Biogenic Amines Proceedings of the First International Workshop Flims, Switzerland February 28-March 7,1987 Editors Η-Ch. Curtius · N. Blau · R. A. Levine

W DE

G Walter de Gruyter · Berlin · New York 1987

Editors Prof. Dr. Hans-Christoph Curtius Dr. Nenad Blau Division of Clinical Chemistry Dept. of Pediatrics University of Zurich Steinwiesstr. 75 CH-8032 Zurich Switzerland Dr. Robert A. Levine Laboratory of Molecular Neurobiology Lafayette Clinic 951 East Lafayette Street Detroit, MI 48207 U.S.A.

Library of Congress Cataloging in Publication Data

Workshop on Unconjugated Pterins and Related Biogenic Amines (1st: 1987 : Flims, Switzerland) Unconjugated Pterins and related biogenic amines. Bibliography: ρ Includes index. 1. Pteridines-Metabolism-Congresses. 2. Biogenic amines-Metabolism-Congresses. 3. Metabolic conjugation-Congresses. I. Curtius, H. Ch. Hans-Christoph) II. Blau, Ν. (Nenad), 1946- . QP801.P69W67 1987 612'.01575 87-22341 ISBN 0-89925-368-7 (U.S.)

CIP-Kurztitelaufnahme der Deutschen Bibliothek

Unconjugated pterins and related biogenic amines : proceedings of the internat. workshop, Flims, Switzerland, February 28 - March 7,1987 / ed. H.-Ch. Curtius ; N. Blau. - Berlin ; New York : de Gruyter, 1987. ISBN 3-11-011341-4 NE: Curtius, Hans-Christoph [Hrsg.]

Copyright © 1987 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: Gerike GmbH, Berlin. Binding: Dieter Mikolai, Berlin. - Printed in Germany.

Dedicated to the Memory of Alois Niederwieser

PREFACE

This volume contains the proceedings of the First Workshop on Unconjugated Pterins

and

Related

Biogenic

Amines,

February 28 to March 7, 1987.

held

in

Flims,

Switzerland,

from

Contributors from all over the world re-

ported on the latest developments in the field of pteridines and biogenic amines.

The workshop brought together pteridine researchers in the fields of chemistry, biochemistry, immunology, pediatrics, and internal medicine from 10 countries; there were 61 participants and 38 papers were presented. Biochemical, main the

analytical,

immunological,

and

themes discussed, but we also heard fields

pteridines.

of

biogenic

amines,

pediatric

problems

were

interesting contributions

chemistry,

and medical

from

applications

of

Each topic was introduced by a plenary lecture.

On February 5, 1987 our colleague and friend, Alois Niederwieser, away

the

after a long

illness.

We dedicate

the proceedings

passed

to him for his

important and lasting contributions in the field of pteridines. Finally we wish to express our gratitude other

participants

who

helped

to

make

to the speakers, chairmen, and the

workshop

a

success;

to

the

authors and all those who helped directly or indirectly with the production of this volume; to M. Killen and M. Stucki for excellent secretarial assistance; to the sponsors, listed separately, for their financial

sup-

port, which made publication of the proceedings possible; and to Walter de Gruyter & Co., for their professional assistance with publication.

H.-Ch. Curtius N. Blau R.A. Levine

ACKNOWLEDGEMENTS We gratefully acknowledge contributions from the following sponsors: Almedica AG, Gal mi ζ, CH ßrechbühler AG, Schlieren, CH Burkard Instrumente AG, Zurich, CH Ciba-Geigy AG, Basel, CH Digitana AG, Horgen, CH Henning Berlin GmbH, Berlin, FRG Hewlett Packard (Schweiz) AG, Widen, CH LKB Instrument AG, Littau, CH Nestle Produkte AG, Zurich, CH Spectronex AG, Basel, CH Technicon Schweiz, Zurich, CH VG Instruments GmbH, Wiesbaden, FRG Milupa AG, Friedrichsdorf, FRG

CONTENTS

In Memorlam: Alois Niederwieser (1935 - 1987) H.-Ch. Curtius

1

SECTION A: CHEMISTRY AND ANALYSIS OF PTERINS Homogeneous Catalytic Hydrogenation of L-Biopterin: Influence of the Ch1ra11ty of the Side Chain and the Catalyst on its Diastereoselective Induction S. Antoulas, M. Viscontini

7

Synthesis of D-Neopterin-3'-Phosphates B. Zagalak, F. Neuheiser, R. Bosshard, U. Redweik

13

5-A1 kyl-5,6,7,8-Tetrahydrobiopteri ns A. Kaiser

23

Mass Spectroraetric Investigations of Pteridines Th. Küster, A. Niederwieser

31

The Structural Characterization of 6-Pyruvoyl Tetrahydropterin by Fast Atom Bombardment Mass Spectrometry with One- and TwoDimensional Mass Analysis (FAB/MS and FAB/MSMS) W.J. Richter, F. Raschdorf, R. Dahinden, Th. Küster, A. Niederwieser, W. Leimbacher, H.-Ch. Curtius

39

χ

SECTION Β: BIOCHEMISTRY AND METABOLISM OF PTERINS

Tetrahydrobiopterin: Other Physiological Roles in Addition to Aromatic Amino Acid Hydroxylation? S. Milstien

49

iH-NMR Studies with Tetrahydrobiopterins, Evidence for the Structure of 6-Pyruvoyl Tetrahydropterin, an Intermediate in the Biosynthesis of Tetrahydrobiopterin S. Ghisla, P. Steinerstauch, Th. Hasler, N. Blau, H.-Ch. Curtius ..

67

Purification of 6-Pyruvoyl Tetrahydropterin Synthase from Salmon Liver Th. Hasler, A. Niederwieser, H.-Ch. Curtius

81

Purification of 6-Pyruvoyl Tetrahydropterin 2'-Keto Reductase from Human Liver H.-Ch. Curtius, P. Steinerstauch, W. Leimbacher, U. Redweik, Sh.-I. Takikawa, S. Ghisla Human GTP Cyclohydrolase I: Monoclonal Antibodies

89

Purification and Preparation of

G. Schoedon, A. Niederwieser, H.-Ch. Curtius

99

Dihydromonapterin Triphosphate: Occurrence, Analysis, and Effect on Tetrahydrobiopterin Biosynthesis in vivo and in vitro N. Blau, P. Steinerstauch, U. Redweik, K. Pfister, G. Schoedon, L. Kierat, A. Niederwieser

105

Biosynthesis of Pteridines and Deaza Flavins in Methanogenic Bacteria B. Schwarzkopf, Q. Le Van, W. Eisenreich, P.J. Keller, H.G. Floss, A. Bacher

117

Tetrahydrobiopterin Biosynthesis and Pteridine Metabolism in Cells of the Blood System, Lymphoid Tissues and in Biological Fluids B. Andondonskaja-Renz, H.-J. Zeitler

125

XI Modified Pteridine Synthesis in Transformed Cell Lines and during Heat Shock J. Seidl, M. Przybylski, Ch. Meinschad, I. Ziegler

137

SECTION C: PTERINS IN IMMUNOLOGY Disease Associated Alterations of Pterin Biosynthesis: Enhancement in Inflammatory Disease, Hemopoietic Regeneration and Endotoxinemla Ch. Huber, M. Herold, J. Troppmair, H. Rokos

149

Metabolism of Pterins in the Cellular Immune System of Man and Mouse G. Schoedon, A. Niederwieser, H.-Ch. Curtius, A. Fontana, J. Troppmair, Ch. Huber

161

Tetrahydrobiopterin Synthesis is Triggered by Interleukin 2 Binding and Modulates Receptor Affinity I. Ziegler

169

Kinetics of Production and Release of Neopterin. Comparison of Human PBMC with the Permanent Monocytic Cell Line U937 and its Subclones K. Rokos, R.O.F. Kunze, Μ.A. Koch, H. Rokos, K. Nilsson

177

SECTION D: PTERINS IN DIFFERENT DISEASES

Variations of Neopterin, Dihydroneopterin and Immunological Parameters during Physical Exercise (Jogging) H. Rokos, K. Rokos, R.O.F. Kunze

187

XII

Biopterin and Neopterln In Plasma and Lympho-Mononuclear Biopterin 1n Graft versus Host Disease and Infection after Bone Marrow Transplantation M. Fink, V. Nüssler, Κ. Demtröder, Ε. Höniges, H.J. Kolb, I. Ziegler

197

Effects of Pirenzepine, Aciclovir and Trimethoprim on Pterins in Blood Cells and Plasma M. Fink, V. Nüssler, Κ. Demtröder, Ε. Höniges, Μ. Goldberg

SECTION Ε: TETRAHYDROBIOPTERIN

207

DEFICIENCIES

Heterogeneity of Tetrahydrobiopterin Deficiency J.-L. Dhondt

219

Prenatal Diagnosis of Tetrahydrobiopterin Deficiency N. Blau, A. Niederwieser, H.-Ch. Curtius, W. Leimbacher, L. Kierat, A. Matasovic, W. Staudenmann, I. Ozalp

237

Sepiapterin Reductase in Human Amniocytes, Skin Fibroblasts, Chorionic Villi and Stimulated Mononuclear Blood Cells J. Ferre, E.W. Naylor

247

Impaired Biopterin Synthesis in a Patient with Mild Hyperphenylalaninemia. A New Variant? J.L. Dhondt, G. Forzy, J.M. Hayte, P. Guibaud, M.O. Rolland, C. Dorche, S. Andre

257

Dihydropteridine Reductase Activity in Fetal Tissues G. Bracco, A. Iavarone, S. Pagliardini, F. Levis, 0. Guardamagna, S. Ferraris, A. Ponzone

265

Molecular Analysis of Human Dihydropteridine Reductase and Dihydropteridine Reductase Deficiency R.G.H. Cotton, W.M. Hutchison, S. Wake, I.G. Jennings, W.J. McAdam, D.M. Danks, Η.-Η.Μ. Dahl

275

XIII Fate of Peripherally Administered Tetrahydrobiopterln 1n Congenital Tetrahydroblopterin Deficiency S. Ferraris, 0. Guardamagna, G. Borietti, G. Bracco, L. Leone, A. Ponzone, A. Niederwieser

283

6-Pyruvoyl-Tetrahydropter1n Synthase Deficiency: Therapeutic Trial with Two Different Synthetic Pterin Analogues in Three Patients D. Leupold, H. Lehmann, H.-Ch. Curtius, A. Niederwieser

293

Placental Barrier in Mother-to-Fetus Transfer of Tetrahydrobiopterin in Humans A. Ponzone, S. Ferraris, S. Biasetti, I. Dianzani, 0. Guardamagna, L. Kierat, A. Niederwieser

303

SECTION F: MONOAMINE NEUROTRANSMITTERS AND ENZYMES Therapeutic Potential of Tetrahydrobiopterin Therapy in Neurological and Psychiatric Illness R.A. Levine

315

Organization of the Catalytic and Regulatory Sites of Rat Liver Phenylalanine Hydroxylase M. Parniak

327

Immunochemical Studies of Phenylalanine Hydroxylase R.G.H. Cotton, I.G. Jennings, W. McAdam, S. Smith

339

Influence of Substrates and Cofactors on Tyrosine Hydroxylase D. Kuhn

343

Mechanism of Activation of Tryptophan 5-Monooxygenase by Mercaptides H. Hasegawa, A. Ichiyama

353

XIV

Effects of Tetrahydropterins on DOPA Production in v i t r o and in vivo M. Bräutigam, W. P f l e i d e r e r Pterins and Monoamine Metabolites in Cerebrospinal

359 Fluid

J . L . Dhondt, G. F o r z y , J.M. Hayte, J . P . F a r r i a u x , C. L a r g i l l i e r e

..

367

AUTHOR INDEX

375

SUBJECT INDEX

379

L I S T OF PARTICIPANTS

393

In Memoriam ALOIS NIEDERWIESER 1935 - 1987

On February 5, 1987 Professor Alois Niederwieser died after a long illness at the age of 52.

We mourn the loss of an outstanding scientist of world

renown, a dear colleague and friend, who shaped to a considerable extent international research in the field of inborn errors of metabolism.

Alois Niederwieser was not only my coworker, he also was a close

friend

for many years and we had been colleagues at the university, thus experiencing a quality of friendship which is not often found in the course of a 1ifetime.

2 Niederwieser came from a practicing catholic family in Laupheim/WLirttemberg and had originally planned to become a priest.

But the life sciences

had caught his interest early and one year before entering university he decided to study chemistry. After two semesters at the University of Tübingen, Niederwieser continued his studies at the renowned Organic Chemistry

Institute of Nobel

prize

winner Tadeus Reichstein in Basle and did his Ph.D. with Max Brenner on thin-layer chromatography of peptides and amino acids. first met Niederwieser.

That is where I

The years of post war reconstruction in Europe

were happy and positive years for all of us. At that time Niederwieser suggested to our future friend, Max Brenner, to separate peptides and amino acids with the help of thin-layer chromatography.

In order to appreciate this proposal one must remember that in

those days chromatography was still in its infancy:

Egon Stahl had just

held a lecture at Roche Company on the separation of pigments by thinlayer chromatography. Organic Chemistry

W.H. Stein, from the USA, had held a lecture at the

Institute on the separation of amino acids by ion ex-

change chromatography, and Max Brenner with his coworker, Rolf Weber, had constructed the first amino acid analyzer in Switzerland according to the specifications by Stein and S. Moore. The first pioneering work on the separation of amino acids and peptides by thin-layer Pataki.

chromatography

was

by

Niederwieser,

Brenner,

and

Four books by Niederwieser and G. Pataki were published, followed

by a number of publications. wieser

published

chamber"

was

Among other inventions the "Brenner-Nieder-

developed which was

still

available

on

the market

until recently. After successfully completing his dissertation, Niederwieser first started work as scientific assistant in the research laboratory of the Neurology Department at the University of Basle.

In 1965 he moved to the University

of Düsseldorf where he worked as research assistant until those years we did not see much of each other. wieser again at the Analytica meeting

1968.

During

By accident I met Nieder-

in Munich and

I aaked him if he

3 would like to join me at the University of Zurich Children's

Hospital.

At that time the new laboratory wing was being completed and we had just started

with

gas

chromatography.

Soon

after

the

Analytica

meeting,

Niederwieser wrote that he would very much like to come to Zurich. In Zurich began.

the most

important 18 years of his fruitful

scientific

work

He published more than 130 papers and was invited to hold more

than 60 lectures which underlined his worldwide reputation as scientist. He discovered and characterized six inborn errors of metabolism, such as ß-mercaptopyruvate

sulfur

transferase

deficiency,

transferase deficiency, 4-hydroxyphenyl

isolated

pyruvate dioxygenase

formimino deficiency,

amino peptidase deficiency, and two enzyme defects of the tetrahydrobiopterin biosynthesis.

In addition, he isolated and elucidated drug metabo-

lites and artefacts of food technology, did analytic pioneering work, such as the separation of peptides as copper complexes, the selective isolation of peptides from biological material by chemical

transformation to poly-

ami no alcohols, GC-MS of oligopeptides in urine, quantitation of mercapturic

acid by mass

fragmentography, measurement of pterins

in urine

by

GC-MS and HPLC, and much more. In the last few years most of his energy was devoted to the pterins. elucidated

two

inborn errors of metabolism, i.e., GTP cyclohydrolase I

deficiency and 6-pyruvoyl tetrahydropterin synthase deficiency. oped a treatment for tetrahydrobiopterin deficiency using pterin

and

He

neurotransmitter

precursors.

From Zurich

He devel-

tetrahydrobio-

he conducted

the

screening for atypical phenylketonuria in many central European countries and overseas. prenatal

Just a short while ago it became possible to perform the

diagnosis of a 6-pyruvoyl

tetrahydropteriη

synthase deficiency.

In the case of an Italian mother who had already given birth to a 6-pyruvoyl

tetrahydropterin

synthase deficient child, Niederwieser performed a

prenatal diagnosis during her second pregnancy which showed normal enzyme activity.

A

healthy

girl

was

born

whom

the

grateful

parents

named

Aloisia, after Alois Niederwieser.

Niederwieser played an important part in the characterization of the most important steps in the tetrahydrobiopterin

biosynthesis.

Recently, to-

4

gether with Wolf Endres pheral

in Munich he proposed that the so-called peri-

defects of 6-pyruvoyl

tetrahydropterin synthase deficiency are to

be found in patients with remaining activity and in heterozygotes. A number of

projects

remain

to be completed.

Niederwieser's

enormous

knowledge and his lucid mind would have facilitated the solving of many more problems. In June of 1986, already marked participated

by

in the 8th International

Acid Derivatives in Montreal.

his terminal

illness,

Niederwieser

Symposium on Pteridines and Folic

As you may have heard, he and I were at

that time nominated to organizethe 9th Symposium in Zurich in 1989. His pioneering work brought him a number of honors. from various

parts of

Many guest scientists

the world visited us in Zurich and

always maintained a warm relationship with them.

Niederwieser

Representative for many

other colleagues in various parts of the world, I would just like to mention the following names: Levine,

Emilio

Oradi,

Martin Gal, Paolo Giliberti, Judy Hammond, Bob Alberto

Pignero,

Haruo

Shintaku,

Shin-Ichiro

Takikawa, Patricia Tippett, and Wang Muti. Niederwieser worked up to the last moment, manuscripts and a book were on his deathbed.

Shortly before he died on February 5 at one o'clock in the

afternoon, he had given instructions to his coworker, Ana Matasovic.

He

died the way he had lived. The

University

of

Zurich

and

the

Children's

Hospital,

pediatricians

throughout the world, and his colleagues have lost a dedicated scientist and a dear friend.

H.-Ch. Curtius

SECTION A: CHEMISTRY AND ANALYSIS OF PTERINS

HOMOGENEOUS CATALYTIC HYDROGENATION OF L-BIOPTERIN: INFLUENCE OF THE CHIRALITY OF THE SIDE CHAIN AND THE CATALYST ON ITS DIASTEREOSELECTIVE INDUCTION

S. Antoulas, M. Viscontini Organisch-chemisches Institut der Universität, Winterthurerstr. 190, CH-8057 Zürich, Schweiz

Introduction Heterogeneous catalytic hydrogenation of L-biopterin (I) under acidic conditions yields a mixture of diastereomers βα-ΐΐ"^ and 60-III in a ratio of approximatively 1/2 [3]. In order to improve this ratio in favour of the 6β-ΙΙΙ isomer (natural form, clinically employed for the treatment of atypical PKU [4]), we tried to hydrogenate the triacetylated derivative IV [5 ] of L-biopterin in presence ot the "Wilkinson catalyst" IX[6].

Η Η I I C-C-CH3 Hzl 1,1 1

0 II

, R2

Η Η ιI

+

OR OR

hAANA l·

CO-CH, = CO-CH,

III : R1 = R2 = Η 1 = CO-CHj ; 2 VI : R R = 1 =2 VIII : R R = CO-CH,

The terms β and α are used according the I UP AC rules for the nomenclature of steroids as propoCI IX, Ph

sed by one of us (M.V.)[1 ] [2 ] .

: Triphenylphosphine

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

8 Even under drastic conditions (60°, 50 At.) no hydrogenation was observed and the substrate could not be recovered from the reaction mixture. On the contrary, the McQuillin catalyst (X)[7] proved to be very efficient. We postulate for this in situ generated catalyst, the following three structures: χ, XI, XII.

ryr-

r

BH4

Vr

Rh r

DMF-

BH4 X

-BH 4

»r

Rh

Rh DMF -

r

CI

DMF-

yr

CI

XI

r

XII

yr

c

·

DMF = Dimethylf ormamide, Pyr = Pyridine, BH^ = Sodium borohydride X and XI are enantiomeric to each other, whereas the third strucsture XII represents a "meso"-form, with a plane of symmetry.

XIV

a=

DMF

b =

EPF*

R VI

1

=

CO-CH

Η

XIII R

3

=

C 1

C 1

1

CH3 1

OR

AC20

Pyr

r VIII

3

Η

OR1 AC2O

XV

Scheme 1

VII

ν

9 We assume that each of the three postulated structures forms a complex with the substrate IV to yield the achiral 7,8-dihydroderivative XIII, which undergoes a second complexation with each of the three structures of the catalyst. In scheme 1 we consider only the second complexation of XIII with the chiral catalyst XI. Depending on wether this new complex formation occurs on the Re(XV) or the Si-side (XIV) of the prochiral C(6) center, hydrogen addition finally gives rise to 60-triacetyl-5,6,7,8-tetrahydro-L-biopterin

(V) and

6a-triacetyl-5,6,7,8.-tetrahydro-L-

biopterin(VI) respectively, which then were converted to the corresponding pentaacetylated derivatives VII and VIII [8]. The approximative 6α/6β ratio was determinated by "^C-NMR spectroscopy, because the spectrum and the absolute configuration of VIII are well known [8]. By comparing the relative intensities of the C(2), C(4) and C(8a) -CO-(CH )

signals, we found that a ratio of ca 1/1 was obtained with McQuillin's catalyst (Figure 1) . C(8a)

In order to get an effective asymmetric induction,

C (4 )

we modified the McQuillin C (2)

α β

catalyst by replacing dime-

βία α 0

ty If ormamide (DMF) with a chiral amide, lR-l-phenyl-

tyVW 13 Figure 1. C-NMR Spectrum of the mixture 6a- 4 6ß-pentaacetyl-tetrahydro-L-biop^^i π i n r n -^n-rn

ethylamino-formamide

(XVIa )

[9] (Figure 2 and 3). The inverse ratio nhfai nod

figure 4. H

C,H.

Figure 2. XVIb = lS-EPF*;(XVIa = 1R-)

With the described experiments we have tried to

10

demonstrate the importance of homogeneous catalysis for the asymmetric hydrogenation of L-biopterin. In order to improve the diastereoselectiv excess -CCMCH )

of the natural 6ß-tetrahydoL-biopterin, other more efficient chirality inducing asymmetric ligands should be developed.

13, Figure 3. Part of the C-NMR spectrum of the mixture VII + VIII after hydrogenation with XVIa (in CD-SO-CD.).

Experimental Part

McQuilling catalyst. According to [7 ] . 1R- and lS-Phenylethylamino-

-CO-(CH 3 )

formamide. According to [9]. Homogeneous Hydrogenation. A solution of lg (2,37 mmol) 2-N-acetyl-l',2'-O-diacetylL-biopterin (IV) in 50 ml CH 3 0H was added to 50 ml C( 8a) CH 3 0H containing 200 mg of in situ generated McQuillin catalyst, and reduced under H^ C(4)

for 3 hours (stirring). The c( 2:

ß

ß p.

reaction mixture was evaporated to dryness and 1,5 ml 12N HCl was added to the residue. The obtained concentred mixture was taken up

13 Figure 4. Part of the C-NMR spectrum of the mixture VII + VIII after hydrogenation with XVIb (in CD 3 SO-CH 3 ) .

in 10-20 ml water and the Rh-complex was extracted twice with 80 ml chloroform.

11

The aqueous phase was evaporated in vacuo and the remaining hydrochlorid was peracetylated under the conditions described in [8]. The crude product was then examinated by "^C-NMR spectroscopy in order to estimate its diastereomeric composition (cf. figures 1,3,4).

References

1.

Ganguly, S.N., Viscontini, M. 1978. Helv. Chim. Acta 61,166.

2.

Antoulas, S., M. Viscontini. 1986. In:Chemistry and Biology of Pteridines (B.A. Cooper, V.M. Whitehead eds.). Walter de Gruyter & Co. Berlin, New York, p. 95.

3.

Schircks, B., J.H. Bieri, M. Viscontini. 1978. Helv. Chim. Acta 61, 2731; S.W. Bailey, J.E. Ayling. 1978. J. Biol. Chem. 253, 1598.

4.

Schaub, J. S. Dämling, H.-Ch. Curtius, A. Niederwieser, K. Bartholome, Μ. Viscontini, Β. Schircks, J.H. Bieri. 1978. Arch, of Diseases in Child. 53, 674; H.-Ch. Curtius, A. Niederwieser, A. Otten, J. Schaub, M. Viscontini, S. Scheibenreiter, Η. Schmidt. 1979. Clin. Chim. Acta 93^ 251.

5.

Furrer, H.J., J.H. Bieri, M. Viscontini. 1979, Helv. Chim. Acta 62_, 2577 .

6.

Osborn, J.Α., F.Η. Jardine, J.F. Young, G. Wilkinson. 1966. J. Chem. Soc.(A) p. 1711.

7.

Love, C.J., F.G. McQuillin. 1973. J. Chem. Soc. Perkins Trans, p. 2509.

8.

Ganguly, S.N., M. Viscontini. 1982. Helv. Chim. Acta 65, 1090; R. Prewo, J.H. Bieri, S.N. Ganguly, M. Viscontini. 1982 . Helv. Chim. Acta 6J5, 1094 .

9.

Kraus, Μ.A. 1973. Synthesis, p. 361.

SYNTHESIS OF D-NEOPTERIN-3"-PHOSPHATES

B. Zagalak, F. Neuheiser, R. Bosshard and U. Redweik University of Zurich, Department of Pediatrics, Division of Clinical Chemistry, Steinwiesstrasse 75, CH-8032 Zurich, Switzerland

Introduction

Recent research in the field of pterins has been focused on the biosynthesis of

(6R)-tetrahydrobiopterin

phosphate

(7,8-H2-3'-NTP).

(BH4)

from

D-7,8-dihydroneopterin-3'-tri-

Obviously, this investigation requires a suit-

able method for the preparation of pure 7.8-H2-3'-NTP.

Enzymatic prepara-

tions of 7,8-H2-3'-NTP obtained from GTP and GTP-cyclohydrolase pure and contain always free GTP and

I are not

7,8-dihydromonapterin-3'-triphosphate

(7,8-H2"MTP) and therefore they cannot be used in a precise

spectroscopic

or enzymatic study.

We have earlier

reported that D-neopterin can be directly

phosphorylated

1

with POCI3 and that D-neopterin-3'-monophosphate

(3 -NMP) was formed

The 3'-NMP was further phosphoryl ated to 3'-NTP.

Unfortunately, we obser-

ved

that a cyclisation

of D-neopterin-3'-monophosphoimidazol idate

(1).

with

a

1

hydroxyl group of the chain takes place after the activation step of 3 -NMP with 1,1 1 -carbonyldiimidazole. low yield

of 3'-NTP.

The

Naturally, this cyclisation causes a very

obtained

3 1 -NTP after catalytic

reduction

to

7,8-H2"NTP was active in the enzymatic synthesis of BH4 (1). Recently we improved the chemical

synthesis of 3'-NMP and 3'-NTP and this

is the subject of this communication.

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

14

Results D-neopterin-3'-monophosphate We found in the course of our further phosphorylation study that preparations of 3'-NMP obtained from direct POCI3 phosphorylation of D-neopterin in tri ethyl phosphate contained up to 15% of unknown D-neopterin monophosphate. This was shown by performing an analytical HPLC on a RP-8 column, using 30 mM Et3N:H3PC>4 buffer, pH 7.0.

The mixture of monophosphates was chromato-

graphically unresolvable at the preparative scale when ion exchangers were used, therefore we gave up this method of phosphorylation of D-neopterin. Recent improvements in the regiospecific synthesis of C-6 substituted pterins and easy

synthesis

of D-ribose-5-monophosphate

gave

us the

idea to

synthesize D-neopterin phosphates via condensation according to the method by Viscontini et al. (2).:

H-C = 0

Η

Η

OH

Η

OH + H 2 N - N - ^

Η

H

OH

-

Η

^ 1 ~C= Ν— Ν

ϊ IV I

+

Η,

R

R

Ο

Ο

Η

,

Η2- C - Ν- Η - P

Η - 4 — O H

CH2OH

Fig. 1

—Φ

Η

C-6-Regiospecific condensation of phenylhydrazones of sugars with 4-hydroxy-2,5,6-triaminopyrimidine.

15 In order to know the pH range of the condensation reactions we began our investigation

with

precise

determination

2,5,6-triaminopyrimidine-2HCl·Η2θ following

pK a

constants:

(7)

and (Τ) 4-OH pK a = 10.076.

(HTAP) 5-NH2

of (Fig.

pK a

pK a

values

2).

= 5.214, ( ? )

We

of

4-hydroxy-

determined N-l

pK a

=

the 1.667

According to Traub & Pfleiderer (3) the pK a

constants of 1, 2 and 3 should be positioned at N-5, N-l and 4-OH respectively.

Fig. 2

Determination of pK a values of 4-hydroxy-2,5,6-triaminopyrimidine-2HCl Ή 2 Ο (2.146mg) using KNO3 (46.6mg) as a standard.

Synthesis de novo via condensation: Adenosine 5'-monophosphate was hydrolyzed with Dowex 50-H + (4) to D-ribose-5-monophosphate which was immediately reacted with Phenylhydrazine at pH 6.2.

Phenylhydrazone of D-ribose-5-

monophosphate was isolated as a barium salt which was further with HTAP in Me0H:H20 (1:1, reflux, 60').

Finally the crude reaction mix-

ture was oxidized with hydrogen peroxide and purified on ion (see

Fig.

10-17%,

3).

The

overall

yield

of

condensed

3'-NMP

tri ethyl ammonium

exchangers salt

was

however according to the HPLC analysis the yield of the condensa-

tion reaction was at least 25-35% (HPLC-assay after the I2:HCl oxidation). Since the triethylammonium

salt is unstable, the end product can be con-

16 Η Η—C = Ο Η·

ΟΗ

Η

ΟΗ

Η

Η—C = Ν - Ν 1.

0 - Ν Η - Ν Η 2 ; lh, RT; pH 6.2

2.

Ba(Ac)2

ΟΗ

Η•

ΟΗ

Η•

ΟΗ

Η

ΟΗ

CH2OPO3H

CH20P03Ba

H s N v ^ A ^ H || Ι *2 H C l η , Ν ^ ν ^ Ν

+

lh, 70°C pH 2.5

Οχ. 1. Lewatit SP 1080 Η+ 2. DEAE-Sephadex; Gradient

OH Η

Fig. 3

2'

Η I ,3' Ο Η

OH — U o I OH

10% yield

Synthesis of D-Neopterin-3'-monophosphate via condensation. (Gradient: Li CI , 0.05-0.4M, pH 7.4 or Et 3 N:H2C03, 0.05-1M, pH 7.6)

17

verted to a stable sodium s a l t by treatment of i t s methanolic solution with an excess of Nal in acetone. this

synthesis

gave

The KMn04 oxidation of 3'-NMP obtained from

pterin-6-carboxylate.

also confirmed by spectroscopic

Structure of

this

31-NMP was

(H-NMR, IR, UV, see F i g . 4) and chromato-

graphic methods. Partial

reduction of t h i s 3'NMP (H2~Pd or sodium hyposulfite) gave 7 , 8 - d i -

hydro-3'-NMP which was identical with enzymatic one obtained from GTP (GTPcyclohydrolase and a l e . phosphatase).

Fig. 4

UV-spectrum of 3'-NMP Na-salt obtained from the condensation.

Synthesis of D-neopterin-3'-triphosphate. a) Enzymatic approach (see F i g s . 5,6):

Guanosine-5'-monophosphate

kinase

(EC 2.7.4.8) catalyses the phosphorylation of GMP using GTP as a phosphate substrate in t h i s system.

The produced GDP i s further phosphory-

lated to GTP in the presence of phosphoenolpyruvate kinase and phosphoenolpyruvate ( 5 ) .

Unfortunately, in our hands t h i s system did not work

when we used 3'-NMP.

Neither did we observe phosphorylation of 3'-NMP

1

to 3'-NDP or 3 -NTP using a similar system with nucleoside-monophosphate kinase (EC 2.7.4.4)

(6).

18

ATP

:

Nucleoside-Monophosphate-Phosphotransferase Ρ

PEP Nucleoside^

Nucleoside^

Nucleoside-Pi ADP

Pyruvate

Nucleoside-Pj-Kinase EC

PEP-Kinase

2.7.4.4

EC 2.7.1.40 (or Nucleoside-5'-P2-Kinase + ATP) 3'-NMP

Fig. 5

(pH 7.4; 37°C)

3'-NDP

Nucleoside-moriophosphate-kinase (EC

ATP

:

2.7.4.4).

(pH 7.4; M g + + ; 37°C)

GMP-Phosphotransferase

PEP GDP

GMP

GTP

ADP

Pyruvate

GHP-Kinase

PEP-Kinase

EC

EC

2.7.4.8 3'-NMP

Fig. 6

-X

Guanosine-5 1 -monophosphate-kinase (EC

2.7.1.40 — 3'NDP

2.7.4.8).

b) Synthesis de novo via the condensation of HTAP with

phenylhydrazone

of D-ribose-5-triphosphate: D-ribose-5-monophosphate monotriethyl ammonium salt was

activated with

phosphorylated

with

l.l'-carbonyldiimidazol

pyrophosphate

(7,8) and

mono-n-tributyl ammonium

yield of D-ribose-5-triphosphate was 60%.

further

salt.

The

This triphosphate was conver-

ted to the phenylhydrazone and further condensed with HTAP. Unfortunately, no 3'-NTP formation was observed (see Fig. 7).

19

H-C = 0 Η — C Η •

O H

Η

O H

Η

O H

H4P20yBU3N

Ο

CH«OP-O 2 I

=0

Η

O H

Η

O H

Η

O H

e

CH2O0

ΟΝ

60% yield

0-NH-NH 2

Ψ H - C = N-N-/0/ Η

O H

Η

O H

Η

OH

NO

CH 2 O(^P 3

g. 7

c)

H

2

N s /

, H

jl j n

TRACE

internal

of protective

groups

MTP

x 2 HCl

Condensation of 4-hydroxy-2,5,6-triaminopyrimidine phenylhydrazone of D-ribose-5-triphosphat.

Inserting

OF

into 3'-NMP:

cyclisation of 3'-NMP-imidazolidate

In order to avoid the

during the

with pyrophosphate, we tried to insert several

with

phosphorylation

O-protective groups into

the chain of 3'-NMP. The

1'-2'-ketalisation

samples

were

refluxed

of

the

3'-NMP

proceeds

with 3 equivalents

with

a good yield

of p-toluenesulfonic

when

acid

in

2,2-dimethoxypropane.

The

1 1 ,2 1 -ethoxyethylether

of 3'-NMP was prepared at 0°-10°C using

vi-

20 nylethyl ether (excess) and 3 equivalents p-tol uenesulfonic acid.

Since

the cleavage of this derivative proceeds relatively fast at pH 4.0 it could be used for the synthesis of 3'-NTP.

On the other hand, we found

the derivatisation procedure (8) to be irreproducible. We have found that 3'-NMP can easily be esterified with 100% formic acid and the produced mono- and diesters can be cleaved under very mild conditions

(pH 8.5-9.0, R.T., 2 h).

This gave us the chance of further

1

phosphorylation of 3'-NMP to 3 -NTP avoiding the cyclisation of 3'-NMPimidazol idate. d) Chemical phosphorylation of 3'-NMP formates to 3'-NTP: is summarized in Fig. 8.

This synthesis

The overall yield of 3'-NTP sodium salt was

10-18%. Preparation of 7,8-dihydro-D-neopterin-3'-triphosphate lithium salt. According

to our experience 7,8-H2~NTP can be easily prepared from much

more stable 3 1 -NTP by partial H2:PdO catalytic reduction at pH 7.0 or using sodium hyposulfite as a reduci ng reagent at pH 6.8. reduction was

terminated

reached 4:1.

The reaction mixture was purified on a DEAE-Sephacel

(9) (see Fig. 9). of

when

In both causes the

the ratio of 7,8-H2-derivatives

to 3'-NTP column

The yield of this step depends on the aerobic conditions

the chromatographic

purification

procedure

and may end up as low as

7,8-^-3'-NTP

preparations was identical

approximate!ly 12%.

The

structure

of both

and con-

firmed by HPLC (RP-8, Et3N:H3P04, pH 7.0 buffer), UV spectrum and kinetics of phosphate release by alcaline phosphatase, when compared with an enzymatically

prepared 7.8-H2-NTP.

Also both preparations obtained by cata-

lytic or hyposulfite reductions were fully active in two different enzymatic systems: the synthesis of BH4 and in the epimerisation of 7,8-H2-3'-NTP to 7,8-H2-3'-MTP.

21

R = Pterin OH

R

•i 1

Η

OH

Η

OH

! 1 Η

f- Ο — l· Η

Ρ = Ο OH

1) HCOOH (100%, RT, 7d) 2) Et3N

c=o i Ο

R

i

c=o Ο

1

1

Η

Η

© ® 0 Et3NH

Η f— 0 Η

OH

1) 2) 3) 4) D-NE0PTERIN-3'-TRIPHOSPHATE·^

Fig. 8

? = 0

60% diester 30% monoesters 10% 3--NMP

H4P20yßU3N, 24h, RT, DMF NaHCOß, H20, 2h, RT DEAE-Sephadex, pH 7.6, gradient NaJ in MeOH/Acetone (10% yield)

One-pot synthesis of D - n e o p t e r i n - 3 ' - t n ' p h o s p h a t .

22

7 , 8 - H z N T P (enzy.)

Fig. 9

Purification of 7.8-H2-NTP on DEAE-Sephacel (1.6 χ 25cm, 0.2M LiCl, flow llml/h, +4°C).

References 1. Zagalak et al. 1985. In: Biochemical & Clinical Aspects of pteridines Vol. 4, (Wächter et al. eds.). W. de Gruyter, Berlin p. 159. 2. Viscontini M. et al.

1970.

Helv. Chim. Acta 53, 1202.

3. Traub H., W. Pfleiderer. 1985. In: Biochemical & Clinical Aspects of pteridines, Vol. 4 (Wächter et al. eds.). W. de Gruyter, Berlin p. 45. 4. Walt D.R. et al. 1984. J. Am. Chem. Soc. 106, 234. 5. Shimono Η., Y. Sugino. 1971.

Eur. J. Biochem. 19, 256.

6. Liebermann I. et al. 1955. J. Biol. Chem. 215, 429. 7. Hoard D.E., D.G. Ott. 1965. J. Am. Chem. Soc. 87, 1785. 8. Kozarich J.W. et al. 1975. Biochemistry 14, 981. 9. Smith G.R., Ch.A. Nichol. 1983. Arch. Biochem. Biophys. 227, 272.

5-ALKYL-5.6.7.8-TETRAHYDROBIOPTERINS

A. Kaiser Pharmaceutical Research Department F. Hoffmann-La Roche & Co. AG CH-4002 Basel

Summary:

Catalytic hydrogenation of 2-N-acyl-l 1 .2 1 -di-O-acylbiopterins and of 1 1 .2'-di-O-acylbiopterins leads to the corresponding 5-alkyl-5,6,7.8-tetrahydrobiopterins hitherto unknown class of compounds.

- a

The mechanism and

the scope of this reductive alkylation and its similarity to the reduction of N(10)-formylfolic acid are being discussed.

During the work on tetrahydrobiopterins that catalytic hydrogenation

it was found

(RT. atmospheric pressure) of

2-N-acetyl- 1',2'-di-O-acetylbiopterin

(1J and

subsequent

acetylation with acetic anhydride/pyridine yielded not only the expected mixture of δα- 1 ^ and 6ß- 2-N. l'-O, 2'-0, 5-tetraacetyl-5.6.7.8-tetra-

hydrobiopterins

(3a. and lb) but

in addition a mixture of 6a- and 6ß-5-ethyl-2-N, 1

l'-O,

2 - Ο , 8-tetraacetyl-5.6.7,8-tetrahydrobio- pterins

(4a^ and

4b). This result was practically independent of the solvents

"^The terms α and β are used for the description of the stereochemistry at C-6 as proposed by Viscontini

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

[1].

24 ( C H 3 C O O H . C F 3 C O O H , M e O H ) u s e d in the

catalytic

h y d r o g e n a t i o n . H y d r o l y s i s of the m i x t u r e of 4a and 4b w i t h a q u e o u s 5N h y d r o c h l o r i c acid gave a m i x t u r e of the 6a- and 6ß-5-ethyl- 5,6,7,8-tetrahydrobiopterins w h i c h one pure d i a s t e r e o i s o m e r

(5a. and 5b).

from

( c o n f i g u r a t i o n at C6 not yet

d e t e r m i n e d ) c o u l d be isolated as its s u l f a t e

(Scheme 1). The

s t r u c t u r e of the 5 - e t h y l a t e d d e r i v a t i v e s 4a^ 4b. 5a^ and E^b 1 13 has b e e n e s t a b l i s h e d by H - and C-NMR-. MS-spectroscopy and e l e m e n t a l a n a l y s i s .

In a d d i t i o n , the

chemical

e v i d e n c e s h o w n b e l o w also points to a 5 - a l k y l a t i o n : a)

C a t a l y t i c h y d r o g e n a t i o n and s u b s e q u e n t a c e t y l a t i o n of 11,2'-O-diacetylbiopterin products

(2_) gave e s s e n t i a l l y the same

(Scheme 1). This result shows that the

N - 2 - a c e t y l g r o u p in the t r i a c e t y l b i o p t e r i n 1. is not i n v o l v e d in the b)

5-alkylation.

C a t a l y t i c h y d r o g e n a t i o n of propionyl-biopterin

2-N-propionyl-l1.2'-di-O-

(6.) f o l l o w e d by h y d r o l y s i s

a q u e o u s 5N h y d r o c h l o r i c acid and s u b s e q u e n t

with

acetylation

w i t h a c e t i c a n h y d r i d e / p y r i - dine lead to a m i x t u r e of the 2 - N . l'-O, 2 1 - 0 , 5 - t e t r a a c e t y l - 5 . 6. 7 , 8 - t e t r a h y d r o b i o p t e r ins 3a. and 3Jb and the 5-propyl-2-N,

l'-O. 2'-0,

8-tetraacetyl-5.6.7,8-tetra-

h y d r o b i o p t e r i n s 7a^ and 7b. H y d r o l y s i s of the m i x t u r e of 7a and 7J3 t h e n y i e l d e d a m i x t u r e of 6a- and 6ß-5-propyl-5,6.7,8-tetrahydrobiopterins f r o m w h i c h a g a i n one pure d i a s t e r e o i s o m e r i s o l a t e d as its sulfate

(Scheme 2). This

(£a and 8b) could be reaction

s e q u e n c e shows that the 5 - a l k y l s u b s t i t u e n t is derived f r o m the l'-0-(or 2'-0-) acyl group and is f o r m e d the c a t a l y t i c c)

during

hydrogenation.

A l l a t t e m p t s failed to h y d r o g e n a t e

the 6a- or

6 ß - t e t r a a c e t y l - t e t r a h y d r o b i o p t e r ins 3a^ and 3J> and the c o r r e s p o n d i n g p e n t a a c e t y l d e r i v a t i v e s £a and

SCHEME

1

OCOCH, OCOCH, 1

^ W C H

ΗΝ

R

—

I CH-CH.

Λ NH R = COCRF-J R = Η H2/PT V

SOLVENT (CH3C0)20 PYRIDINE

0

FOCH3

' \

^

Ν

^ Ν Η

3A

CH7

OCOCH3OCOCH3 —

CH-CH3

FH2

+

OCOCH, OCOCH, ! •> I -> 'CH -CH—CH,

CH3COH ( A COCH3

(6>)

3b ( 6 3 )

MA

(6Α)

4B

(SP)

5Ν HCL

AQ

CH, FH2

OH

YN

N\J;H-CH-CH

5A (.6S)

5b (6R)

3

26 SCHEME

2

OR OR I I N^/CH—CH-CH

6

R =

3

C0CH2CH3 H2/PT

-J' S O L V E N T I 5N H C L

AQ.

(CH3C0)20 ^

fH3

PYRIDINE

CH-, 0

CHJCOH

C O C H , ? C 0 C H 3 OCOCH3

11 j NH

^ H

tH—CL·

+

IH2 ΗΝ

9 C O C H 3 OCOCRL-J £H

CH—CR

CH3C0N! COCH-

3a

(6a)

7a

(6a)

3b

(6e)

7b

(68)

r

5N H C L

AQ.

CH, I 5 CH2 ^ < J H - C H - C H

5

27 respectively

(Scheme 3). indicating that the formation of

these products during the hydrogenation of 1 and 2 is not the reason for the formation of 5-ethylated

products.

SCHEME 3

2a

R = Η 9α R = C0CH3

(6α)

^

R = Η 9b R = C0CH3

(6ß)

h2/Pt

V NO

CF7COOH 3

REACTION

The results described above point out that the formation of 5-alkylated products during the catalytic 1

of 1',2 -di-O-acylbiopterins

hydrogenation

can best be explained by the

intermediate formation of an oxazolinium compound B) which is then hydrogenated

(e.g. A or

to the 5-alkyl derivative.

Theoretically, this oxazolinium compound can be formed either from N(5)- or from 1 1 -O-acylated

tetrahydrobiopterins

(e.g. C or D ) (Scheme 4). All attempts to isolate acyl derivatives of these structures in pure form have not met with success.

28

SCHEME 4

Η

Η

C

D

Preliminary results indicate that the catalytic hydrogenation described above is applicable to a broad range of 1',2'-di-O-acyl-biopterins and thus constitutes the first general synthesis of the hitherto unknown 5-alkyl-5.6.7.8tetrahydrobiopterins. Moreover, the reaction sequence Ε—>F—>G

(Scheme 5) seems to be applicable to other

6-hydroxyalkylpteridines as well. Finally, the similarity of the hydrogenation of O-acylated biopterins to the reduction of N(10)-formylfolic acid should be mentioned. The question arises whether reaction of O-acylated biopterin and other

this

6-hydroxyalkyl

substituted pteridines or their tetrahydro derivatives is also of biological

significance.

29 SCHEME 5

Ε

0

OCOR'

F R'

G

Acknowledgement The help of Ms. N. Erhardt, Dr. U. Fischer. Ms. S. Herzig, Ms. C. Piers, Dr. W. Arnold. Dr. A. Dirscherl, Dr. E. Englert, Mr. W. Meister and Dr. W. Vetter is gratefullyacknowledged.

Reference

M. Viscontini 1985. In: Biological and Clinical Aspects of Pteridines (W. Wächter. Η. Ch. Curtius, W. Pf leiderer, eds.) Walter de Gruyter u. Co.. Berlin. New York. p. 57

MASS

Th.

SPECTROMETRIC

Küster,

Department CH-8032

A. of

INVESTIGATIONS

OF

PTERIDINES

Niederwieser Pediatrics,

Zurich,

University

of

Zurich,

Switzerland

INTRODUCTION

Due

to

field

the

of

lyzing

increasing

biochemistry

this

microbiological

for

routine

The on

hand

compounds

very

present

or

well here

mental

in

high

details

see

been

performance

however, many

cases.

to

developped. are

of

from

our

of

the ana-

Whereas suited

chromatography (1,2).

chromatographic

methods

identification

Mass

such

mainly

in

results

spectrometry

chromatography

solve

examples

methods

liquid

prevents

gas

gained

quantitative

selectivity

suited

various

radioimmunoassays

without

some

have

promising

poor

other

combination be

very

inherent

known

and

pteridines

medicine,

class

analysis,

yields

the

and

compound

the

(HPLC)

interest

(GC)

problems

un-

(MS)

in

arises

to

(3-7)

laboratory.

of

For

and

we

experi-

(6-8).

RESULTS

GC-MS

For

the

trace it

in

ments, a

identification

analysis, our for

control

GC-MS

laboratory the for

of is for

unknown

the the

method

routine

of

choice

monitoring

characterization

HPLC

compounds

of

new

of

as

well

and

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

for

we

applied

loading

experi-

metabolites

determinations.

as

and

as

32

a ) Loading

experiments

Atypical

forms

of

a

BH^,

barrier.

tutes

which

patient

served 1

can

enter

lites found

only

the

the

more

easily

with

doses

the

blood

passes

find

cofactor

than

before

excreted

and

and

after

loaded

and

loading.

identified

in urine samples during

substi-

BH^, we

6-methyltetrahydrobiopterin

pterin

that laoading

obTable

metabo-

test.

Identified Pterins

Time after loading(hrs)

Biopterin Neopterin 6-Methylpterin 6-Methylisoxanthopterin + + +

+ + + +

Excreted

pterins

before 2-4 4-6 12-24

-

Table

slightly to

pterins

mainly

successfully

efforts

brain

with

excreted

the

be treated

which

During

orally

the

shows

PKU

cofactor

brain

a

of

1:

-

after

+ +

+ +

+

+

loading

with

6-Methyltetra-

hydrobiopterin.

Fig.

1

shows

the

excretion

6-methyl-isoxanthopterin value 4

of

hours

slower than

212

and

after

of

pterin

due

to

excreted

mainly

is produced

(from

from

unchanged

During

tests

the

by

HPLC

recovery

resting

reached

between

shows

to

a

whereas

for

the

lies

in

maximal 2 and

a

25

fact

the measured

to

0.7)

that

is

6-methylwhich

was

6-methylisoxanthopterin oxidase.

metabolites usually

occurence the

and

significantly

(from

0.3),

that

by the action of xanthine

loading

The

6-methyltetrahydropterin

of new

gated

212

circumstance

b) Characterization BH^

neopterin.

is

figure

6-methylpterin

6-methylisoxanthopterin

the

originates

The

of

to

respectively

6-methy lpterin

possibly

relative

loading.

decrease

of

25

pattern

region

urine

samples

of pterin of

are

investi-

metabolites.

1-5% only,

if these metabolites are also excreted

it

was

in the

Since inte-

feces.

33

100-

10-

hrs.

2-4 Figure

1: Total

τ

4—β

12—24

ion current peak area r a t i o s

Methylisoxanthopterin +

to Neopterin

of

6-Methylpterin and 6-

versus time a f t e r

loading

with 6-Methyltetrahydropterin. Figure feces the C

2

shows

fraction.

sample

has

a

total

For

the

been

ion

current

determination

spiked

with

even

chromatogram of

of

the methylene

numbered

such a units,

n-alkanes

C101ο

26 "

RIC.

2Θ0

Figure

250

2:

Total

ion

3Θ0

8:20

6:40

5:00

current

chromatogram

10:00 of

a feces

350 11:40 fraction

34 Table

2

lists

BH^treatment All

the

identified

together

with

their

listed

have

been

compounds

references

lumazines

and

their

methylene

in

feces

respective verified

units

Compound

after

methylene

by

the

units.

comparison

with

(8).

No .

MU

Μ

Base

'(%)

TMS

peak

6-Hydroxylumazine

3

20 .61

396(100)

7,8-Dihydro-6-hydroxylumazine

4

21 . 5 5

470(100)

2 '-Deoxy-biolumazine

3

21 .67

438(

90) 409

7,8-Dihydro-2*-deoxybiolumazine

4

22 .36

512(

40) 422

2'-Deoxysepialumazine

3

22 . 72

438(100)

Biolumazine

4

23 .07

526(0.6)

7 ,8-Dihydrobiolumazine

5

23 .85

600(100)

Sepialumazine

4

2 3 .93

526(100)

2 '-Deoxysepialumazine

4

24 .67

510(100)

Table

2:

Lumazines

identified

in

feces

after

tetrahydrobiopterin . MU : methylene lecular

c)

Control

The

amount

urine In

for

question

ble

results

we

do

with a

this a

L-threo-neopterin

could

arises An by

of

proof

pterin

is

of

units,

+

M ' :

trimethylsilyl

value

the

0.2 that

the

of mo-

groups.,

with

excreted

is

3

two

HPLC

to

observed of

a reference

really

shows of

monapterin

neopterin. HPLC the

peak

methylene

compound.

and

yields

then

relia-

indicated

and

chromatograms (A)

GC-MS 2

in

(neopterin).

ratio

then

value

comparison

method

this

control

Figure

relative

exeeds

HPLC

independent

excretion

(monapterin)

D-erythro-neopterin

the

if

GC-MS.

through

spectrum

10%

however

normal

ratio

mass

of

cases

the

of

ingestion

HPLC

normally

some

ion,No.TMS:number

410

and

Β

here

really units

with

firstly is and

monathe

34 ι

Fluorescence 350/450 nm

1

U

Β

A

min 3:

HPLC

pterins to

cation

of

bration tive

values

value

Direct

HPLC

determined

0.21 of

excreted for

for

the

ion

monapterin. and

The

the

urine

for

mentioned

Chemical

Ionization

(DCI)

compounds

with

components

a

less

its

advantages

relative volatile

high which

without

decomposition.

As we

pterins

can

volatile

and, which gated

this

made

method

especially does

more

not

therefore

for

results

the

it

4

a

mark

reslults

for

mixture

are

is

pterins

by

gives "true".

fails

be

of with

vaporized TMS-ethers, but

than

determination desirable.

HPLC

analysis

derivatization,

weight

respec-

GC-MS

the

questions

cali-

Thus,

it

cannot

the

the

B.

HPLC

more

2

quantifi-

shows

whereas

with

mona-

biopterin.

sample

here

high

neopterin,

4 =

arrows

often

molecule

reference

(B)

volatility,

by

in

molecular

change some

did

and

allowed

ratio

the

30

=

Figure

GC-MS.

that

has

1

monitoring

indicating

GC-MS

normal

ratio.

0.22

Whereas

times,

(A)

3 = isoxanthopterin,

selected

the

plots

yielded a

of

with

neopterin

monapterin,

method

min

30 0

of

pterin

The

2

3

,0 Figure

35

We

direct

some-

answers a

method

investichemical

36

Figure 4: Ratio determination of Monapterin to Neopterin by HPLC(·--—•) and GC-MS(# # ) . Ratio values determined, ( • ) using the corresponding c a l i b r a t i o n graph: HPLC.0.21, GCMS.0.22.

ionization,

a technique

and

so

can

be

usually

is

a

evaporated high

molecular

weight.

determines

the

intensity So,

3 lists

the

the

gated

pterins

and

pterin-6-carboxylic limits

quasi

from d e c a r b o x y l a t e d

ions

the

5

in

protonated

molecular

acid. acid

of

sample

shows

This ion rather

the

The

result

region

molecular

of

ion

the often

weight. ions

(MH+)

the

DCI

example

also

at

is flash vaporized

decomposition.

molecular

Figure

since

the

without

respective

Table

method's

by which

m/z

than

of

mass

the

investi-

spectrum

shows some o f

164 a r i s e s from the

to

intact

of the

originate molecule

37

Compound

MG

MH+

(%)

Lumazine 6-Hydroxylumazine Pterin Pterin-6-carboxylic acid Xanthopterin Isoxanthopterin Sepiapterin Biopterin Neopterin Monapterin

164 180 163 207 179 179 235 237 253 253

165 181 164 208 180 180 236 238 254 254

(100) (100) (100) ( 27) (100) (100) ( 48) (100) (100) ( 75)

Base peak

164

103

103

Table 3: Molecular weights (MG), Quasimolecular ions (MH ) and base peaks of reference pterins under direct chemical ionization conditions with methane as reactant gas (9).

100

-

75

-

164

190

50 UJ >

147 25

-

94

122

r^U, f M r

80

130

208

MH*

Jvi I .1 • , 180

230

m/z

Figure 5: DCI (methane) spectrum of pterin-6-carboxylic acid (9).

" r ~T~ r 280

38 ACKNOWLEDGEMENT

We

express

Houston,

our

thanks

Texas,

USA

for

to

Prof.

the

E.C.

Horning, University

of

DCI-analyses.

REFERENCES 1.

Fukushima, 176 .

T.,

J.C.

Nixon.

1980.

2.

Niederwieser, a . , W. S t a u d e n m a n n , Ε . W e t z e l . 1982. In: B i o c h e m i c a l a n d C l i n i c a l A s p e c t s of P t e r i d i n e s ( H . W ä c h t e r , H . C h . C u r t i u s a n d W. P f l e i d e r e r , e d s . ) de G r u y t e r , B e r l i n , I, p. 8 1 .

3. K o b a y a s h i , Κ . , M . G o t o . 1 9 7 0 . of P t e r i d i n e s (M. G o t o a n d Y. P r i n t i n g C o . , T o k y o , p. 5 7 . 4. L l o y d , T . , 108. 5. R ö t h l e r , 6.

S.

F.,

Markey,

Μ.

N.

Karobath.

Anal.

Biochem.

102,

In: C h e m i s t r y and Biology Iwanami, eds.) Int. Acad.

Weinen.

1976.

1971.

Clin.

Anal.

Chim.

Biochem.

Acta

69_,

42,

457.

Küster, 245.

Th. ,

A.

Niederwieser.

1983.

J.

Chromatogr.

2 78,

7. K ü s t e r , 303.

Th. ,

A,

Niederwieser.

1984.

J.

Chromatogr.

290,

8. N i e d e r w i e s e r , Α . , A. M a t a s o v i c , T h . K ü s t e r , W . S t a u d e n m a n n W. P f l e i d e r e r , S. S c h e i b e n r e i t e r . 1 9 8 6 . I n : C h e m i s t r y a n d B i o l o g y of P t e r i d i n e s (B.A. C o o p e r , V.M. W h i t e h e a d , e d s . ) de G r u y t e r , B e r l i n , p. 3 0 5 . 9. H o r n i n g , ration .

E.C.,

Η.-Ch.

Curtius,

A. N i e d e r w i e s e r :

in

prepa-

THE STRUCTURAL CHARACTERIZATION OF 6-PYRUVOYL TETRAHYDROPTERIN BY FAST ATOM BOMBARDMENT MASS SPECTROMETRY WITH ONE- AND TWO-DIMENSIONAL MASS ANALYSIS (FAB/MS AND FAB/MSMS)

W.J. Richter, F. Raschdorf, R. Dahinden Central

Function Research, Ciba-Geigy AG, CH-4002 Basel, Switzerland

A. Niederwieser+, H.-Ch. Curtius, W. Leimbacher, Th. Küster University of Zurich, Department of Pediatrics, Division of Clinical Chemistry, CH-8032 Zurich, Switzerland

Introduction

Tetrahydrobiopterin important

role

(BH4),

in the

the cofactor

biosynthesis

of various

hydroxylases,

of the neurotransmitters

plays

an

catecholamine

and indoleamine (1), and inborn errors in the BH4 biosynthesis cause atypical phenylketonuria.

Clarifying the biosynthetic pathway (Scheme 1) there-

fore is of great importance for the elucidation of metabolic defects. key

intermediate

hydropterin mode

of

(2-6).

hereby

(PPH4)

formation,

is

a compound

structure.

This

its chemical

with

a

hypothesis

proposed has

6-pyruvoyl

been

deduced

reactivity, and from trapping

The

tetra-

from

its

experiments

Whereas all these experiments led to indirect evidence for the PPH4

structure, Ghisla et al. (7) presented direct ^H-NMR evidence for the pyruvoyl

tetrahydro

structure.

In order

to

have

further

direct evidence

we

present here mass spectrometric results toward the structure elucidation of the

intermediate.

Results and Discussion

The in

intermediate Scheme

bombardment

2)

had (FAB)

in the been mass

BH4 biosynthesis, inferred,

was

spectrometry.

for which a PPH4 structure

subjected A

to

useful

analysis

by

positive

ion

Unconjugated Fterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

fast

{Αχ, atom

spectrum

40

Ο

^

OHOH

F ^ - C - C H j - O ® ® ® NhUTP H

HjN^N^V Η

0

Η

^ B '

0 OH

0 Mg2+

V.

Η

NAD(P)H

0

Η

xXT

OHO

Η

'PPH^S"

h

0 0 ii ii

Hv

^B-v

PPH A (A t )

c-c-ch3

Η 2, κ τ ν Ν τ Η tSR)

0

VH

NAD(P)H

•'PPH4R"

Η

Ηι II0 C-C-CH3 1 OH η2Ν-%"%Τ * Η

0

ύΥΤ

ί

Η

Ο Η

ΟΗ Η

SR>>NAD(P)H

NAOfPJH-^^SR γ 9

Π Η ι-Ηι Η

ΗΝ

ΒΗ> N"Sr

OHOH

Scheme 1 : Biochemical pathway of BH4 biosynthesis from dihydroneopterin triphosphate. (Fig. 1) was acquired using thioglycerol

(glySH) as the liquid matrix, in

which a [M + H] + ion of moderate abundance was observed at m/z 238 (ca. 20% rel. int.). elemental

This

indicated the presence of a molecule with the correct

composition

C9H11N5O3

(base peak) was attributed

(M=237).

to a [M + H]

The +

intense

signal

at m/z

ion of 7,8-dihydropterin

166

(PH2,

C6H7N5O, M=165) which was formed by decomposition of the moderately stable new compound either before or during analysis. the

formation

protonated

of the m/z 166 ion by genuine

An alternative route for gas phase

fragmentation

of

PPH4, e.g., via loss of 0=C=C(0H)CH3 from an intact [M + H] +

41

precursor,

appeared

less

probable

importance

in the metastable

since

such

a process

is of only

ion (MI) decomposition which may be

minor

observed

by FAB/MSMS (see below).

Scheme 2. Structures considered as the intermediate in tetrahydrobiopterin biosynthesis: Al, 6-pyruvoyl tetrahydropterin; A2, sepiapterin; A 3 , biopterin; A4, A5, see text.

The

(+)FAB behavior of known isomers and hydrogenate analogues of

different matrices was of special

interest in this context.

mers, the most important ones, i.e., sepiapterin were readily available spectrum in a glycerol

for a preliminary

1

study .

[M +

Difficulties H]

+

A2 yielded a useful

(glyOH) matrix displaying a [M + H ]

formation

were

however

required

the

encountered

use

(A3),

(A2) and biopterin +

with

A3,

in

Of these iso-

FAB

ion at m/z 238,

ca. 15% rel. int., together with other major ions of the (glyOH) n type.

PPH4

as

of m-nitrobenzylalcohol

even

cluster modest

(ΝΟΒΑ)

as a

1) A more detailed study of the FAB behavior of pteridines is currently in progress (W.J. Richter, F. Raschdorf, Th. Küster, and H.-Ch. Curtius).

42

liquid

matrix

(8)

since

t h e 10% r e l .

int.

signal

at

a "chemical

noise"

l e v e l o f up t o 8% appeared t o be u s e l e s s u n l e s s MS-MS was employed.

While

t h e A4 and A5 isomers were n o t o b t a i n a b l e , t h e h i g h l y u n s t a b l e analogue 0 - or p - q u i n o n o i d d i h y d r o b i o p t e r i n f o r comparison.

As e x p e c t e d ,

hydrogenated

( q u i n o n o i d BH2) was

available

t h i s analogue (M=239) a l s o proved

difficult

t o a n a l y z e ( g l y S H m a t r i x ) , as an e f f i c i e n t uptake o f 2 Η atoms was observed which p r o b a b l y y i e l d s 1:5). the

The a n a l y s i s (+)FAB

spectrum

BH4 as t h e s t a b l e

product

(m/z 2 4 0 / 2 4 2 r a t i o

about

o f BH4 (M=241) d i d however prove t o be s u c c e s s f u l (glySH m a t r i x )

showed e x c l u s i v e

[M +

H]+

as

formation

( m / z 2 4 2 , base p e a k ) .

10 Ch

166

/

50-

(M+H) + 238

I···"!' .1.11.1 ·ί ι. • • I. i.l • ι. • ] 1 i·. ι. ι .Iii 11 ii •• • .1•-ιlillllllllMilillilillllliilllillillllll mtii mhln.iiiilll m/z

140

—

180

220

F i g . 1 . (+)FAB spectrum o f the i n t e r m e d i a t e A^ ( t h i o g l y c e r o l 10 keV xenon a t o m s ) . From F i g .

1 and t h e

preliminary

results

,III,ill

obtained with

analogues i t i s q u i t e obvious t h a t s t r u c t u r a l

matrix,

these

isomers

and

p a r a m e t e r s , o t h e r than molec-

u l a r s i z e , c a n n o t be d i r e c t l y deduced from FAB/MS, owing t o a l a c k o f p r o -

43 nounced FAB

fragmentation

in conjunction

of

with

a clear-cut origin. two-dimensional

mass

Under

these

spectrometry

circumstances, (FAB/MSMS)

peared highly promising since the ions of interest, i.e., the [M + H]

+

apions

of intact PPH4, can be singled out for further MS analysis as more or less pure

species.

The "dynamic", i.e., nonchromatographic

ions, that

is, separation

prevailing

"chemical

from other

ions

noise", is accomplished

the employed tandem mass spectrometer.

such

as

"isolation" of the

those

of

PH2 and

in the first stage, MS-I

permits

of

MS-II then performs the mass analy-

sis of the daughter ions which are generated in a collision cell between MS-I and MS-II.

the

positioned

Operation of the cell with or without a target gas

the options of collision

induced dissociation

(CID) and

spontane-

ous, i.e, metastable ion (MI) decomposition, respectively.

m/z 238

Fig. 2. Mass analyzed ion kinetic energy (MIKE), FAB/MSMS spectra of [M + H] + ions (m/z 238) of the intermediate Aj (_a, upper trace), sepiapterin (b, center trace) and biopterin (c, lower trace).

44

F i g . 2 shows the FAB/MSMS spectra of the relevant mass-selected [M + H] + The [M + H] + ion of the

i o n s , m/z 238, which were recorded in the MI mode.

"unknown", which i s to be compared with those of the reference ions derived from ß>2 and A3, shares a pronounced l o s s of H2O with i t s isomers. case of

structure A^, which lacks a hydroxy!

substituent as a preformed

leaving group, a rearrangement by hydride s h i f t ( s ) of H2O from protonated ketones see ( 9 ) ) .

In the

i s indicated (for

loss

The pronounced elimination of a

42-Dalton neutral, which i s most l i k e l y to be CH2=C=0 from the terminus of the side-chain,

seems to be highly s p e c i f i c

for the new d e r i v a t i v e .

The

genesis of the corresponding daughter ion at m/z 196 may a l s o be r a t i o n a l ized by assuming that a f a s t 1,2-hydride [M + H]

+

species

occurs

as the i n i t i a l

shift step

in a carbonyl-protonated

(Scheme 3 ) .

The

rearranged

species, i . e . , the product of an intramolecular "redox" process leading to a dihydropterin,

should permit a "Grob-type"

fragmentation which produces

m/z 166 Scheme 3. Proposed mechanism f o r CH3C(0H)=C=0 from [M + H] + of PPH4.

the

elimination

of

CH2=C=0

and

45 an acetyl cation CH3C=0 + together with a neutral hydroxymethylenamine molecule.

The high proton affinity of the neutral may favor subsequent proton

abstraction from CH3CsO +

(the potential

Bronsted acid) prior to dissocia-

tion of the intermediate ion/dipole complex, thereby rendering CH2=C=0 the final neutral ejected.

It seems plausible that the di- and nonhydrogenated

pterins such as A2 and A3 do not lend themselves readily to analogous reaction pathways. of

The "compound specificity" of the MI daughter ion spectrum

the [M + H]

+

of

the new derivative

existence of the new isomer, but also

supports

not only the

its isolation

without

postulated large-scale

decomposition by rearrangement into A2 or A3 during the workup and/or FAB analysis. As mentioned above, the formation of a protonated PH2 (m/z 166) by MI ejection of CH3C(0H)=C=0, rather than chemical decomposition, is inconspicuous. It should nevertheless be considered, as it places the additional

oxygen

1

atom clearly into the C(1 )-carbonyl group of the assumed CH3COCO substituent.

The specificity of this latter process

is unfortunately

not suffi-

ciently well established owing to the low relative intensity and broadness of the m/z 166 MI signal

(see e.g. A2).

The MS evidence for a tetrahydro-

genated pterin nucleus is therefore insufficient.

When specificity is only admitted for the MI elimination of ketene from the C3

substituent

in a di- or tetrahydrogenated

assignment of Αχ has still these

alternatives

quinonoid

0-

or

p-tautomer

pterin via an endiol

structural

two major competitors, i.e., A4 and A5.

represent

1'-hydroxy-2'-oxopropyl

pterin skeleton,

dihydropterins, 6,7-dihydro

substituent.

i.e.,

a 7,8-dihydro-

derivative

carrying

a

A 4 , which is tautomeric with

Both and

a

common sepia-

intermediate, is ruled out because of its chromophore

which is incompatible with the observed UV absorption.

The

UV-compatible

chromophore A5 is a highly unlikely competitor owing to its FAB/MS behavior which

sharply

contrasts

that

of

0-

or

p-quinonoid

6,7-dihydrobiopterin

since the quinonoid BH2 reference compound A3 rapidly takes up 2 Η atoms prior to or during FAB analysis.

In the case of the new derivative no such

hydrogen uptake was observed (Fig. 1). derived

from

other

spectroscopic

The PPH4 structure Αχ, which was

evidence

without

actual

isolation,

therefore complemented and fully supported by mass spectrometrie means.

is

SECTION Β: BIOCHEMISTRY AND METABOLISM OF PTERINS

TETRAHYDROBIOPTERIN: OTHER PHYSIOLOGICAL ROLES IN ADDITION TO AROMATIC AMINO ACID HYDROXYLATION? Sheldon Milstien, Ph.D. Laboratory of Neurochemistry National Institute of Mental Health Bethesda, MD 20892

Introduction

The two major unconjugated pterins present in human serum, urine and cerebrospinal fluid are dihydroneopterin and tetrahydrobiopterin (BH^) (1).

The only established physiological role for BH^ is as the natural

cofactor in the hydroxylation of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan (2).

BH^ is used by the three different enzymes

which catalyze the hydroxylation reactions in conjunction with enzyme-bound iron to reduce molecular oxygen (2).

A genetic defect in

phenylalanine hydroxylase blocks the metabolism of phenylalanine and causes phenylketonuria (3).

Children with genetic defects in either de

novo BH^ biosynthesis or in dihydropteridine reductase, the enzyme responsible for maintaining BH^ in its reduced form, have both an inability to metabolize phenylalanine, as well as a lack of the important neurotransmitters, dopamine, epinephrine, and serotonin, which are derived from the hydroxylated amino acids (A).

The origin of dihydroneopterin in human tissues has not yet been

This paper is dedicated to the memory of Dr. Alois Niederwieser

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

50

unequivocally established.

It probably arises as a result of

dephosphorylation of dihydroneopterin-triphosphate, the first pterin derivative in the de novo BH^ pathway (5).

The measurement of

dihydroneopterin levels in blood and urine has proven to be a useful clinical parameter in cancer (6), AIDS (7), and organ transplantation (8). Dramatic increases in neopterin excretion are seen whenever lymphoid cells, such as Τ cells or macrophages, are stimulated by interferon or IL-2 (9).

The role of dihydroneopterin in this or any other physiological

process has as yet not been established.

Clearly, dihydroneopterin cannot

be an indispensable factor, since: many species have essentially no neopterin in their body fluids or tissues (1); many human cells, such as fibroblasts, do not produce any neopterin; and children with a genetic block in GTP-cyclohydrolase, the enzyme which catalyzes the formation of dihydroneopterin triphosphate, have no apparent anomalies in their immune systems (10).

The presence of BH^ in many tissues and in brain regions nearly devoid of any aromatic amino acid hydroxylase activity (1), raises the possibility that BH^ might also be necessary for the activity of a different kind of enzyme system.

Several other oxidation reactions have been proposed to

utilize a tetrahydropterin as a cofactor.

These include: ether lipid

oxidation (11); proline hydroxylation (12); and mitochondrial electron transport (13).

The evidence for the requirement for a tetrahydropterin

in these enzyme systems is reviewed below.

In addition, as a guide to the

discovery of other roles for BH^ and it's biosynthetic enzymes, the significance of the discovery that dihydropteridine reductase, an enzyme

51 which had previously been thought to only be involved in maintaining BH^ in the fully reduced state, also plays an important role in tetrahydrofolate metabolism, is discussed.

Distribution of BH^ and Its Biosynthetic Enzymes in Rat Brain

BH, is present in every region of the rat brain (1A) and the human brain (15) with a fairly narrow concentration distribution.

Since the only

known function of BH^ in the CNS is to serve as a cofactor for tryptophan and tyrosine hydroxylases, it is not surprising that a correlation has been found between the activity of these enzymes in various regions of the rat brain and the concentration of BH^ (1A).

Using an enzymatic cofactor

assay, Levine, et al (1A), showed that there was a high degree of correlation (r = 0.8) between reduced cofactor content and total tryptophan plus tyrosine hydroxylase activities in the rat brain.

In

Figure 1, are plotted BH^ concentrations, determined by a high performance liquid chromatographic assay (1), against the total tryptophan and tyrosine hydroxylase activities in seven areas of the rat brain.

There is

a high degree of correlation between BH^ content and hydroxylase activities, even though the HPLC method gives somewhat greater values than the cofactor assay.

It should be noted that there is more than a 120 fold

range in the hydroxylase activities between the cerebellum and the striatum, whereas there is only a 5 fold range in BH^ concentration. difference in the ranges suggests that if the BH^ in the striatum is concentrated almost solely in aminergic neurons as has been suggested (16), this cannot be the case in other areas of the brain since the concentration of BH, would be extremely high in those areas with only

This

52

relatively few aminergic neurons, such as the cerebellum and cortex. Also, extrapolation of the data in Figure 1 to a hypothetical region of the brain with no hydroxylase activities indicates that there would still be significant amounts of BH^ remaining.

Another approach which can be used to evaluate the localization of BH^ and its biosynthetic enzymes in specific aminergic neurons is the application of neurotoxins, such as 6-hydroxydopamine, 5,7-dihydroxytryptamine, kainic acid, and MPTP.

These toxins are taken up by receptor-mediated processes

and then kill those cells which concentrate them by still not well characterized mechanisms.

6-Hydroxydopamine and MPTP are taken up

selectively by cells in the substantia nigra dopaminergic pathway and cause symptoms in animals similar to those of Parkinson's Disease in

Μ

1 2 3 4 5 6 ?

60τ

striatum cerebellum medulla htypothalumus hippocampus cortex midbrain

~ B0+ (P J 40+ 2 ο I ο

±

30-. 20+

a

ίοt 0

lfc

2

ια

•ι— 4• f—

•σ r— Ο ΜΙ

ί .

•

CSJ •

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CO

LO π-

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αι

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ο

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ο ο —

CL

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ϊ

I

•

•—•

c Γ— Γ0 4-> Ο I—

*

>>

+->

ί .

.—•

ε

*

Ο 05

— , -—»

ε «•Μ*

LO ΙΟ Ο

—

ο ο Γ—

•

ο ο ΙΟ

ο ιο r—

ο

— ,

•

CO •

ο

«3-

Ο

LO CO I

CSJ csj I

CSJ

αϊ i . Ζ3

Ό Φ υ ο s_

+J Ο A3 S-. +J Χ αι αι •σ ίΟ

(β 4t— 3 (Λ ε •r—

C ο ε

C ο • r— +J Ο (β S4S« Ο LO I Ο

αι -Μ •Γ—

re

+->

ο. > χ ο iΤ3 >5

υ 1

αι

13 CQ

ι 0Ω

+->

95 After Blue Sepharose a f f i n i t y chromatography the increase of s p e c i f i c tivity

was about 550-fold.

This

purification

ac-

factor does not take

into

account the f i r s t two p u r i f i c a t i o n steps which lead to a 100-fold p u r i f i c a t i o n , approximately.

The total amount of protein was

human l i v e r corresponding to an overall

recovery of 28%.

Sepharose step some contaminating proteins were s t i l l in

Fig.

1).

Mono Q ionic

single

After the Blue

present (see SDS-PAGE

These were eliminated by threefold FPLC chromatography

on

exchanger.

at

This

resulted

in

two f r a c t i o n s

280 nm), both of them showing PPH4R a c t i v i t y . still

0.3 mg from 300 g of

contaminated

with minor

band corresponding

protein

While the f i r s t

impurities,

to a molecular

(detection

the

peak was

second y i e l d e d a

weight of approx. 35ΌΟΟ

D on

66 kDa

US kDa 56 kDa 29 kDa 2t kDa 20 kDa

-

ο ο

14 kDa

in Οι

F i g . 2. SDS-Page with 12.5% Polyacrylamide. Left gel: enzyme preparation from d i f f e r e n t p u r i f i c a t i o n steps, as mentioned in the f i g u r e ; r i g h t g e l : active fractions from the t h i r d Mono Q run. The mixture of standard proteins contained: bovine albumine (66 kD), egg albumine (435 kD), g l y c e r i n aldehyde-3-phosphate dehydrogenase (36 kD), carbonic anhydrase (29 kD), trypsinogen (24 kD), t r y p s i n i n h i b i t o r (20 kD), a-1actalbumin (14 kD).

96 SDS-PAGE (Fig. 2).

Since this corresponds to the molecular weight deter-

mined by using gel

filtration, it must be concluded that the enzyme is a

monomer. Analytical

isoelectric

focusing

(pH range 4-9, Servalyt Τ 4-9) on G-200

superfine revealed an isoelectric point of pH 6.0 ±0.2.

Glycoprotein de-

tection using Concanavalin A and peroxidase was negative.

In the presence

of 10 mM DTE less activity was found than with a 1 mM solution, suggesting that PPH4R is not particularly sensitive to -SH group oxidation.

Only PPH4

and not 6-(Γ-oxo-2'-hydroxypropyl)-5,6,7,8-tetrahydropterin or sepiapterin is a substrate for this enzyme. 5,6,7,8-tetrahydropterin

will

Most probably 6-(r-hydroxy-2'-oxopropyl)also

turn out to be a substrate

for this

enzyme, however, so far this has not been investigated in detail. indicates a much higher catalyzing

This

specificity of PPH4R compared to SR, the latter

the reduction of a variety of diketo functions

(25).

It is,

therefore, reasonable to assume that this new enzyme indeed plays a role in BH4 biosynthesis, although this still remains to be proven by direct experiments.

A particular problem is posed by nomenclature. hydropterin 6-pyruvoyl

reductase"

might

not

be

The name "6-pyruvoyl tetra-

appropriate,

since

SR also

reduces

tetrahydropterin, although with a different preference for the

side chain carbonyl

groups.

"6-pyruvoyl

tetrahydropterin 2'-keto

reduc-

tase" or "6-pyruvoyl tetrahydropterin 2'-carbonyl reductase" might be better choices. The use of specific inhibitors for PPH4R and of antibodies which are currently

in preparation might help to elucidate

the question whether

this

enzyme is on the biosynthetic pathway or not, and to solve the sequence problem mentioned above.

97 C o n d usion An enzyme which catalyzes the reduction of the

2'-keto group of 6-pyruvoyl

tetrahydropterin, the intermediate in tetrahydrobiopterin biosynthesis, has been purified from human liver to apparent homogeneity.

The enzyme has a

native molecular weight of approx. 3 5 Ό 0 0 D and is a monomer.

It appar-

ently contains no carbohydrates and has an isoelectric point of pH 6.0.

In

the presence of NADPH this enzyme efficiently catalyzes the formation of 6-(r-oxo-2'-hydroxypropyl)-5,6,7,8-tetrahydropterin hydropterin.

from 6-pyruvoyl tetra-

1

6-(l -oxo-2'-hydroxypropyl)-5,6,7,8-tetrahydropterin

and

sepiapterin are no substrates.

Acknowledgements This work was supported by the Swiss National no. 3.395-0.86.

We are grateful

Science Foundation, project

to Dr. B. Mermuth for providing us with

the carbonyl reductase.

References 1.

Curtius, H.-Ch., S. Takikawa, A. Niederwieser, S. Ghisla. 1986. In: Chemistry and Biology of Pteridines 1986 (B.A. Cooper and V.M. Whitehead, eds.). de Gruyter, Berlin, p. 141.

2.

Curtius, H.-Ch., D. Heintel, S. Ghisla, Th. Küster, W. Leimbacher, A. Niederwieser. 1985. J. Inher. Metab. Dis. 8 Suppl. 1, 28.

3.

Switchenko, A.C., G.M. Brown. 1985. J. Biol. Chem. 260, 2945.

4.

Milstien, S., S. Kaufman. 1099.

5.

Masada, M., M. Akino, T. Sueoka, S. Katoh. 1985. Biochim. Biophys. Acta 840, 235.

6.

Nie hol, C.A., G.K. Smith, D.S. Duch. 1985. Ann. Rev. Biochem. 54, 729.

7.

Blau, Ν., A. Niederwieser. 1983. Anal. Biochem. 128, 446.

1985. Biochem. Biophys. Res. Commun.

128,

98 8.

Ghisla, S., P. Steinerstauch, Th. Hasler, N. Blau, H.-Ch. Curtius. 1987. In: Unconjugated Pterins and Related Biogenic Amines. (H.-Ch. Curtius and N. Blau, eds.). de Gruyter, Berlin (this volume).

9.

Richter, W.ü., F. Raschdorf, R. Dahinden, A. Niederwieser, H.-Ch. Curtius, W. Leimbacher, Th. Küster. 1987. In: Unconjugated Pterins and Related Biogenic Amines. (H.-Ch. Curtius and N. Blau, eds.). de Gruyter, Berlin (this volume).

10. Switchenko, A.C., J.P. Primus, G.M. Brown. 1984. Biochem. Biophys. Res. Commun. 120, 754. 11. Smith, G.K., C.A. 761.

Nichol.

1984. Biochem. Biophys. Res. Commun. 120,

12. Smith, G.K., C.A. Nichol. 1985. J. Biol. Chem. 261, 2725. 13. Takikawa, S., H.-Ch. Curtius, U. Redweik, S. Ghisla. 1986. Biophys. Res. Commun. 134, 646.

Biochem.

14. Takikawa, S., H.-Ch. Curtius, U. Redweik, W. Leimbacher, S. Ghisla. 1986. Eur. J. Biochem. 161, 295. 15. Milstien, S., S. Kaufman.

888.

1983. Biochem. Biophys. Res. Commun.

115,

16. Katoh, S., T. Sueoka, S. Yamada. 1982. Biochem. Biophys. Res. Commun. 105, 75. 17. Heintel, D., W. Leimbacher, U. Redweik, B. Zagalak, H.-Ch. Curtius. 1985. Biochem. Biophys. Res. Commun. 127, 213. 18. Wermuth, B. 1981. J. Biol. Chem. 256, 1206. 19. Hasler, Th., A. Niederwieser, H.-Ch. Curtius. 1987. In: Unconjugated Pterins and Related Biogenic Amines. (H.-Ch. Curtius and N. Blau, eds.). de Gruyter, Berlin (this volume). 20. Laemmli, U.K. 1970. Nature, London 227, 680. 21. Clegg, J.C.S. 1982. Anal. Biochem. 127, 389. 22. Radola, B.J. 1974. Biochim. Biophys. Acta 386, 181. 23. Katoh, S. 1971. Arch. Biochem. Biophys. 146, 202. 24. Niederwieser, Α., W. Staudenmann, E. Wetzel. 1984. J. Chromatogr. 290, 237. 25. Sueoka, T., S. Katoh. 1985. Biochim. Biophys. Acta 843, 193.

HUMAN GTP CYCLOHYDROLASE

I:

PURIFICATION

AND PREPARATION

OF MONOCLONAL

ANTIBODIES *

G. Schoedon, A. Niederwieser+ , H.-Ch. Curtius D i v i s i o n of C l i n i c a l Chemistry, Department of P e d i a t r i c s , U n i v e r s i t y of Zurich, CH-8032 Zurich, Switzerland

Introduction The f i r s t

step

in

the biosynthesis

of

tetrahydrobiopterin

(BH4)

is

the

conversion of GTP to dihydroneopterin triphosphate (NH2TP) and formic acid, which i s catalyzed by the enzyme GTP cyclohydrolase I (GTPCH; EC 3 . 5 . 4 . 1 6 ) . This enzyme has f i r s t been characterized from Escherichia c o l i ( 1 , 2 ) , l a t e r its

purification

from several

other microorganisms

(3,4,5),

insects

(6),

chicken ( 7 ) , rat (8) and, p a r t i a l l y , from human (9) has been described. GTP cyclohydrol ase I deficiency in man causes atypical to the lack of BH4 b i o s y n t h e s i s

and deficiency

of

phenylketonuria due

the

neurotransmitters

dopamine and serotonin (10). Immune stimulation causes a d r a s t i c a l of

GTPCH a c t i v i t y

in the responsible c e l l s

increase

of both man and mouse

(11).

These findings gave the reason for further studies of the human GTPCH on the molecular and gene l e v e l , including p u r i f i c a t i o n and s t a b i l i z a t i o n the enzyme as well as production of monoclonal

of

antibodies.

Two c h a r a c t e r ! s t i e s of human GTPCH made i t s p u r i f i c a t i o n e s p e c i a l l y

diffi-

c u l t : One i s the f a i l u r e of a f f i n i t y chromatography for the mammalian enzyme and the other first

is

its

extreme i n s t a b i l i t y when beeing p u r i f i e d . The

has been overcome by development of a new a f f i n i t y material

8-aminoguanosine

triphosphate

and

sepharose

4B,

which

resulted

using in

a

300-fold p u r i f i c a t i o n (12). But due to the i n s t a b i l i t y of the enzyme, only 5% of a c t i v i t y has been recovered.

* This paper i s dedicated to the memory of Prof. A l o i s Niederwieser

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

100 The present paper describes a p u r i f i c a t i o n

procedure of GTPCH from human

l i v e r modified with respect to the s t a b i l i z a t i o n of enzyme a c t i v i t y .

Materials and Methods All

chemicals

procedure

for

used are commercially GTPCH w i l l

available.

be reported elsewhere

The detailed (13).

purification

GTPCH a c t i v i t y was

measured as described in (9). Proteins were determined by the dye binding method {Bio Rad). Production of monoclonal antibodies has been done according to Köhler and M i l s t i e n ELISA and Western b l o t t i n g .

(14). Monoclonal

antibodies were selected by

Isotyping was performed by use of

Ig-subclass

s p e c i f i c double sandwich ELISA (Bio Rad, subisotyping k i t ) .

Results and Discussion Based on the repeated observation that GTPCH a c t i v i t y i s unstable in d i l u t e solution,

studies

of

the conditions

under which the a c t i v i t y

unstable or stable have been made. For t h i s ,

is

either

GTPCH a c t i v i t y was measured

after storage for 24 h at 4°C, -20°C or -70°C in l i v e r homogenate as well as in diluted

samples

in d i f f e r e n t

buffers

with and without

stabilizing

agents. Generally,

all

diluted

fractions

tested were very unstable,

freezing

of

these samples at -20°C eliminated even more a c t i v i t y than storage at 4°C. Lyophilization of the samples was not successful. The loss of a c t i v i t y the diluted

samples was p a r t i a l l y

avoided only by the addition of

in

large

amounts of bovine serum albumin, but not by DTE or 2-mercaptoethanol to the buffer.

In homogenate, the enzyme was found to be more stable in phosphate

buffer than in T r i s , therefore a 50 mM potassium phosphate buffer at pH 7.8 was chosen as basis solution for GTPCH extraction. Addition of glycerol to this buffer even increased the s t a b i l i t y of the enzyme, but did not cause an a l t e r a t i o n of a c t i v i t y . Almost no loss of a c t i v i t y was found in the ammonium sulfate fraction, and, when i t was necessary to store the enzyme between p u r i f i c a t i o n steps, the best way was p r e c i p i t a t i o n and storage of the p e l l e t at 0-4°C.

101

Additionally,

highly diluted samples were found to be f u l l y active

after

storage as a suspension in Sephadex G-200 superfine ( s f ) at 4°C.The a c t i v i ty from aliquots of t h i s suspension could e a s i l y be eluted over small G-25 columns. S l u r r i e s stored

of p u r i f i e d enzyme in G-200 sf and 10% glycerol

can be

several days at 0-4°C without l o s s of a c t i v i t y .

For longer time, rapid concentration and storage in 50% glycerol at -70°C was found to counter the l o s s of a c t i v i t y at l e a s t for 3 months. Since i t became cl ear that GTPCH s t a b i l i t y

i s dependent on concentration

and protein environment, and that l o s s of enzyme a c t i v i t y

i s a factor of

time, a p u r i f i c a t i o n procedure had to be developped, which i s f a s t , keeps the enzyme as concentrated as p o s s i b l e ,

avoids

freezing

steps or

longer

storage periods, and in which the main s t a b i l i z e r s , ammonium s u l f a t e ,

gly-

cerol and superfine dextran p a r t i c l e s , can be used. Thus, human l i v e r GTPCH was p u r i f i e d by 1. extraction with 50 mM phosphate b u f f e r , pH 7,8, containing 5% g l y c e r o l , 2. ammonium s u l f a t e

precipitation

between 30% and 40% saturation, 3. chromatography on Ultrogel

AcA 34 and

f i n a l l y , preparative i s o e l e c t r i c focussing on Sephadex G-200 superfine. The f i r s t two steps can be done in 1 day from a large batch of l i v e r , and the 40% ammonium sulfate p e l l e t can be stored at 4°C overnight or, i f not the whole batch i s processed further, a l i q u o t s can be stored at -20°C. Step 3 has the advantage that no desalting step ( d i a l y s i s , G-25) which causes l o s s of a c t i v i t y i s needed for the processing of the ammonium sulfate

fraction.

The enzyme i s eluted from the AcA 34 column in phosphate buffer containing 25% glycerol

and i s

running as a sharp peak j u s t behind the void volume.

This makes separation f a s t and removes proteins of lower molecular weight nearly t o t a l l y .

The AcA 34 pool containing GTPCH i s concentrated r a p i d l y

on an Amicon PM-10 u l t r a f i I t e r and a s l u r r y i s made of 50 mg Sephadex G-200 sf/ml protein s o l u t i o n . The s i u r r y can be stored at 4°C until i t i s aplied on a horizontal

preparative

isoelectric

focussing

gel

made of

Sephadex

G-200 s f , 10% (v/v) g l y c e r o l , and 2% (v/v) ampholine Τ 4-9. The dimension of the gel layer i s dependent on the amount of protein in the mixture, the loading capacity beeing 5-10 mg protein per ml gel suspension. GTPCH focusses at pH 5,6 =pl with high recovery of a c t i v i t y .

102

A purification

summary

of the GTPCH is given in Table 1. Analysis of the

fractions by SDS-polyacrylamid gel electrophoresis showed reduction of the complex the

protein mixture to a few bands. The major band is enriched during

purification

procedure,

beeing

a

protein

of

50Ό00

dal tons.

By

gel

filtration, an apparent molecular mass of 4 4 0 Ό 0 0 dal tons was estimated for the active enzyme. Thus, human GTPCH is a complex of 8 subunits.

Table 1

Purification of GTP-Cyclohydrolase I from Human Liver

Procedure

Total protein

Crude extract Ammonium sulfate

Total units

Specific activity

Yield

Purification

uU/mg prot.

%

fold

mg

uU

57 050

44 000

0.7

100

1

5 240

38 800

7.4

88

10

1 100

32 600

30.0

74

43

18 980

9 040.0

43

13 300

30-40 % Ultrogel AcA 34 IEF G-200 sf/

2.1

G-25 m pool

GTPCH I is purified from 300 g of human liver.

After isoelectric focussing, the enzyme is

eluted from the G-200 gel over Sephadex G-25 m, concentrated, substituted Hith glycerol to 50 X (v/v), and stored at -70°C.

The activity Is stable for 3 months.

1 uU = lpmol NTP/min at 37°C.

Recently, we were able to produce monoclonal antibodies against human GTPCH. For immunization the protein isolated by isoelectric 1

focussing was

used. Mixed with complete Freund s adjuvant, 20 ug were injected neously in each of 4 Balb/c mice. One month after the primary

subcuta-

immunization,

antigen in incomplete Freund's adjuvant was injected. Serum obtained after two additional

immunizations was tested for activity of specific antibodies

by Ouchterlony double diffusion and ELISA. The two mice with highest titers were

boosted with

days before well

antigen

fusion. The

as cloning

in 0.9%

NaCl

intraperitoneal ly one week and 3

fusion was achieved

in HT-medium

in PEG, and HAT-selection

by use of the limited dilution method

as

were

performed. Eleven monoclonal antibodies were obtained, two of IgM subclass, the others IgG^.

103

In ELISA against homogenous GTPCH from E.coli, 4 of the monoclonals showed activity. In western blotting, they bound against the 50'000 dal ton protein of the human enzyme. With these antibodies, an immunocytochemical method is under

investigation

to localize

staining

GTPCH in the stimulated

human

macrophages and T-cells. They may also be useful for enzyme studies on the molecular and gene level.

Acknowledgement This work was supported by the Swiss National

Science Foundation, project

No. 3.601-0.84 and No. 3.395-0.86. We are grateful Zurich, for the possibility to perform monoclonal

to Dr. H. Hengartner, antibody production in

his laboratory and Mr. U. Redweik for excellent technical assistance during isoelectric focussing experiments.

References 1.

Burg, A.W., G.M. Brown. 1968. J. Biol. Chem. 243, 2349.

2.

Yim, I.I., G.M. Brown. 1976. J. Biol. Chem. 251^, 5087.

3.

Cone, I.E., I. Plowman, G. Gurolf. 1974. J. Biol. Chem. 249, 5551.

4.

Jackson, R.J., T. Shiota. 1975. Biochim. Biophys. Acta 403, 232.

5.

Kohashi, Μ., T. Itadani, K. Iwai. 1980. Agric. Biol. Chem. 44, 271.

6.

Weisberg, Ε.P., J.Μ. 0'Donnell. 1986. J. Biol. Chem. 26^, 1453.

7.

Fukushima, K., W.E. Richter, Τ. Shiota. 1977. J. Biol. Chem. 252, 5750.

8.

Bellahsere, Z., J.L. Dhondt, J.P. Farriaux. 1984. Biochem. J. 217, 59.

9.

Blau, Ν., A. Niederwieser. 1983. Anal. Biochem. 128, 446.

10.

Niederwieser, Α., Ν. Blau, Μ. Wang, Ρ. Joller, Μ. Atares, J. GardesceGarcia. 1984. Anal. Biochem. 141, 208.

104

11.

Schoedon, G., J . Troppmair, A. Fontana, Ch. Huber, H.-Ch. A. Niederwieser: Eur. J . Biochem. ( i n p r e s s )

Curtius,

12.

B l a u , Ν., A. Niederwieser. 1986. Biochim. Biophys. Acta 880, 26.

13.

Schoedon, G., A. Niederwieser, H.-Ch. C u r t i u s : et al .: i n

14.

K ö h l e r , G., C. M i l s t i e n . 1975. Nature (Cond.) 256, 495.

preparation

DIHYDROMONAPTERIN TRIPHOSPHATE: OCCURRENCE, ANALYSIS, AND EFFECT O N T E T R A H Y D R O B I O P T E R I N B I O S Y N T H E S I S in vivo AND in vitro

N.Blau, P.Steinerstauch, U.Redweik, L.Kierat and A.Niederwieser+

K.Pfister,

G.Schoedon,

D i v i s i o n of Clinical Chemistry, Department of Pediatrics, U n i v e r s i t y of Zürich, CH-8032 Zürich, S w i t z e r l a n d

SUMMARY 6 - P y r u v o y l - t e t r a h y d r o p t e r i n synthase (PPH4S) f r o m h u m a n and salmon liver is inhibited by L-monapterin (L-threo-neopterin). D - M o n a p t e r i n as well as d i h y d r o and t e t r a h y d r o - L - m o n a p t e r i n h a v e no e f f e c t on PPH4S. Lowered biopterin excretion as well as induction of neopterin excretion in u r i n e of mice was o b s e r v e d after t r e a t m e n t with L - m o n a p t e r i n . Incubation of m y e l o m a cells w i t h L-monapterin indicates t h a t a p t e r i n d e f i c i e n c y can be induced in v i v o by inhibition of PPH4S. L-Monapterin is formed from dihydromonapterin triphosphate (MHaTP) after dephosphorylation and oxidation. MH2TP produced from dihydroneopterin triphosphate (NH2TP) by dihydroneopterin triphosphate 2 ' - e p i m e r a s e was separated as monapterin t r i p h o s p h a t e (MTP) by HPLC. Dihydroneopterin triphosphate 2'-epimerase from E. coli was p u r i f i e d b y ammonium sulfate precipitation, DEAE-Sephadex A25 chromatography, Sephadex G 1 5 0 gel f i l t r a t i o n and isoelectrofocusing on S e p h a d e x G200 SF.

INTRODUCTION L-Monapterin is the major pterin found in some microorganisms. R e m b o l d and Heinz (1) isolated this compound from cell lysates of E s c h e r i c h i a coli in 1971 b u t its

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

106 biological function is still unknown. The function of m o n a p t e r i n in C o m a m o n a s sp. w a s p r o p o s e d b y G u r o f f and R h o d a s t o b e t h a t of a c o f a c t o r of p h e n y l a l a n i n e hydroxylase (2). They a l s o d e s c r i b e d the occurrence of a dihydroform of m o n a p t e r i n in t h i s bacteria. U r u s h i b a r a et al. (3) suggested t h a t t h e c y c l i c m o n o p h o s p h a t e of m o n a p t e r i n m i g h t b e i n v o l v e d in m e t h a n o l oxidation by a non-specific primary alcohol dehydrogenase

in

Bacterium

methylococcus

capsulatus.

M o n a p t e r i n h a s b e e n r e p o r t e d t o occur a l s o in S e r r a t i a i n d i c a (4) and in P s e u d o m o n a s roseus fluorescens (5), however in n e i t h e r r e p o r t t h e b i o l o g i c a l s i g n i f i c a n c e w a s stated. A n u m b e r of a u t h o r s (6,7,8) s u g g e s t e d t h a t t h e c y c l i z a t i o n of D-erythro-neopterin monophosphate to 2', 3 ' - c y c l i c - n e o p t e r i n monophosphate in some m i c r o o r g a n i s m s may result in an i n v e r s i o n of t h e 2 ' - h y d r o x y 1 g r o u p of t h e p r o p y l side chain of n e o p t e r i n t o y i e l d t h e L - t h r e o epimer. However, later on it w a s v e r i f i e d t h a t d i h y d r o n e o p t e r i n triphosphate is the initial pterin formed from GTP and t h a t the initially r e p o r t e d c y c l i c m o n o p h o s p h a t e of n e o p t e r i n a p p e a r s t o b e an artifact produced during purification procedures when an a l k a l i n e s o l v e n t is used. A n i n t e r e s t i n g p o i n t is t h e o c c u r r e n c e of m o n a p t e r i n in h u m a n tissues and urine but neither the biosynthesis nor its b i o l o g i c a l f u n c t i o n in m a m m a l i a n s was explained. Monapterin like n e o p t e r i n in u r i n e is m o s t l y p r e s e n t in its d i h y d r o form and t h e r a t i o of m o n a p t e r i n t o n e o p t e r i n is u s u a l l y about 1:10. In d i f f e r e n t b l o o d cells this r a t i o v a r i e s from 1:10 to 1:3 (9). H o w e v e r , w e h a v e n o p r o o f t h a t its f o r m a t i o n occurs n o n e n z y m a t i c a l l y f r o m d i h y d r o n e o p t e r i n or e n z y m a t i c a l l y from dihydroneopterin triphosphate. We just have some observations that monapterin may possibly be formed from d i h y d r o n e o p t e r i n if u n t r e a t e d u r i n e s are s t o r e d for a longer p e r i o d of time. Furthermore, w e n e v e r n o t i c e d t h a t m o n a p t e r i n w a s f o r m e d e n z y m a t i c a l l y in a n y m a m m a l i a n m a t e r i a l . Ten y e a r s ago, H e i n e and B r o w n (10) d e s c r i b e d in E. coli an enzyme, named D-erythro-dihydroneopterin triphosphate 2'-epimerase, that catalyzes the c o n v e r s i o n of N H ^ T P to

107

MHaTP. They were able to show that, all three phosphate residues of the substrate are retained in the product and that the product is of the L-threo configuration but they were not able to separate the phosphorylated product from the substrate chromatographically. Recently, we noticed that different preparations of NH2TP which we use as a substrate in the PPH4S assay show different activities. We were surprised that some preparations of NH 2 TP, which we synthesized enzymatically from GTP by GTP cyclohydrolase I, contained up to 50% MH 2 TP. In this paper we describe the separation of M H a T P from N H 2 T P by HPLC after oxidation with iodine, as well as the effect of M H 2 T P and its metabolites on BH4 biosynthesis in vitro and in vivo. The purification of N H 2 T P 2'-epimerase from E. coli is also described.

MATERIALS AND METHODS Most of the pterins were purchased from Dr. B. Schircks Laboratories, 8645 Jona, Switzerland. D-Monapterin was synthesized by Prof. M. Viscontini, and dihydroand tetrahydro monapterin were prepared by reduction with Pd/H 2 . N H 2 T P was synthesized with GTP cyclohydrolase I immobilized on Sepharose 4B (11). Measurement of PPH4S and sepiapterin reductase activities were performed as described earlier (12). The HPLC system for measurement of pterins was the same as described elsewere (13). Isoelectrofocusing was performed on Desaga flat bed electrophoresis unit (Heidelberg, GFR). Gel (8 mm thickness) consists of Sephadex G200 SF with 10% glycerol and 2% Servalyt T4-9. Electrofocusing was performed at 5 W for 100 min and at 7 W for 170 min at 3"C. 5-mm slices of the gel were assayed for enzyme activity. HPLC of neopterin and monapterin phosphates The HPLC analytical procedure was essentially as described earlier (14), except that the RCM system with Guard Pak C18 + Nova Pak C18 5ju (5 χ 100 mm) from Waters was 'used.

108 I s o p r o p a n o l in m o b i l e p h a s e w a s o m i t t e d a n d f l u o r e s c e n c e m e a s u r e d on a H i t a c h i F 1000 f l u o r o m e t e r . A s s a y for N H 2 T P

was

2'-epimerase

T h e a s s a y for N H 2 T P 2 ' - e p i m e r a s e a c t i v i t y w a s based on the H P L C m e a s u r e m e n t of M T P ( o x i d a t i o n p r o d u c t of MH2TP) formed f r o m N H 2 T P . The p r o c e d u r e of H e i n e and B r o w n (10) was u s e d t o generate M H 2 T P from NH2TP. The reaction mixture (100 |ul) c o n t a i n e d 25 μ Μ N H 2 T P , 5 m M M g C l a , 1.4 m M EDTA, 0.1 g/1 BSA, and 50 jul e n z y m e preparation in 25 m M 2-(N-morpholine) e t h a n e s u l f o n i c acid b u f f e r p H 6.25. The r e a c t i o n p r o c e e d e d at 3 7 e C for 15 m i n in t h e d a r k and w a s terminated by the a d d i t i o n of 10 jul of a 1 Μ HCl s o l u t i o n c o n t a i n i n g 130 iodine and 2% KI. A f t e r s t a n d i n g at r o o m t e m p e r a t u r e for 15 min, p r o t e i n s w e r e r e m o v e d b y c e n t r i f u g a t i o n and e x c e s s iodine w a s r e d u c e d b y a d d i t i o n of 10 μ ΐ 1% a s c o r b i c acid. The c o n t e n t of M T P w a s a n a l y z e d in 5 /al of s u p e r n a t a n t b y HPLC. The volume of t h e e n z y m e p r e p a r a t i o n in t h e a s s a y m i x t u r e was c h o s e n so that the ratio MTP/NTP at t h e end of incubation never e x c e e d e d 1.0. P u r i f i c a t i o n of N H 2 T P

2'-epimerase

The enzyme was purified from Escherichia coli using m o d i f i c a t i o n s of t h e p r o c e d u r e of H e i n e a n d B r o w n (10). The protein precipitate obtained after fractionation between 50 and 8035 a m m o n i u m sulfate was dissolved in 60 ml 50 mM p o t a s s i u m p h o s p h a t e buffer, pH 8.0. The concentrated and dialyzed enzyme preparation was applied to a DEAE-Sephadex A 2 5 c o l u m n (5 χ 30 cm) equilibrated with 0.1 Μ Tris-HCl buffer, pH 8.0 (starting buffer). NHaTP 2'-epimerase was e l u t e d w i t h 2 0 0 0 ml of a linear g r a d i e n t t o 0.5 Μ LiCl in t h e s a m e b u f f e r at a flow rate of 100 ml/h. F r a c t i o n s b e t w e e n 520 and 9 6 0 ml containing NH2TP 2'-epimerase activity were pooled, c o n c e n t r a t e d t o 16 ml on an A m i c o n Y M - 1 0 u l t r a f i l t e r , and a p p l i e d t o a Sephadex G150 column (2.6 χ 100 cm) e q u i l i b r a t e d w i t h 0.1 Μ p o t a s s i u m p h o s p h a t e buffer, pH 8.0. T h e p r o t e i n s w e r e e l u t e d w i t h t h e same b u f f e r at a flow rate of 10 m l / h and 5 ml f r a c t i o n s w e r e collected. The fractions

109

53 to 66 with NHaTP 2'-epimerase activity were combined and concentrated to a volume of 5.8 ml. An aliquot of the fraction (2.5 ml) was concentrated to a volume of 0.9 ml on an Amicon YM-10 ultrafilter and applied to a Sephadex G200 SF gel prepared for isoelectrofocusing as described above. The gel slide at 8 to 9 cm (pH 7.0 - 6.4) was taken out and 2 ml of 1 Μ NaCl were added. The enzyme was eluted from gel with 0. 5 Μ NaCl in 10 mM potassium phosphate buffer, pH 7.0 and washed from ampholyte on a Sephadex G25m column (1.6 χ 26 cm) equilibrated with 10 mM potassium phosphate buffer, pH 7.0. The eluate was concentrated on an Amicon YM-10 ultrafilter to a volume of 2.8 ml and stored at -20°C.

RESULTS AND DISCUSSION We modified our HPLC system for the separation of neopterin phosphates and guanine nucleotides so that we were able to separate neopterin phosphates from monapterin phosphates (Fig. 1).

1 I

0

2 |V.

Η 10

L· ain

Fig. 1 HPLC of neopterin and monapterin phosphates l=neopterin, 2=monapterin, 3=NMP, 4=MMP, 5=NDP, 6=MDP, 7=NTP, 8=MTP Omission of isopropanol in the mobile phase improved the separation of isomers and use of the shorter column enabled the separation in less than 20 min. However, separation of

110

d i h y d r o f o r m s of n e o p t e r i n and m o n a p t e r i n t r i p h o s p h a t e is not p o s s i b l e w i t h this system. Neopterin and monapterin t r i p h o s p h a t e o b t a i n e d after incubation of N H 2 T P with NH2TP 2'-epimerase and s u b s e q u e n t oxidation w i t h iodine were h y d r o l y z e d at room t e m p e r a t u r e to the c o r r e s p o n d i n g d i - , and m o n o p h o s p h a t e s y i e l d i n g finally n e o p t e r i n and m o n a p t e r i n as hydrolysis p r o d u c t s (Fig. 2). α min

b min

Ι-

-Λ

20 nln

10 mir

V

60 nln

80 min

V| 1\ -.-/Λ...

Fig.2 Hydrolysis of neopterin and m o n a p t e r i n trip h o s p h a t e w i t h alkaline phosphatase. Numbers of the peaks refer to compounds in Fig.1 M H z T P was d e t e c t e d after incubation of GTP w i t h different preparations of G T P cyclohydrolase I from E. coli. P r e p a r a t i o n s of N H 2 T P containing d i f f e r e n t amounts of MH2TP were u s e d in the PPH4S assay and found to p r o d u c e variable results. These observations suggested t h a t MH?TP or one of its metabolites may effect the tetrahydrobiopterin biosynthesis. To find out at w h i c h step the inhibition occurs we started a n u m b e r of experiments by incubating monapterin and its reduced forms w i t h enzymes involved in the biosynthesis of tetrahydrobiopterin. Incubations with enzymes p u r i f i e d from h u m a n and salmon liver showed t h a t monapterin but n o t d i h y d r o m o n a p t e r i n and t e t r a h y d r o m o n a p t e r i n inhibit the a c t i v i t y of PPH4S (Tab. 1). P P H 4 S from salmon liver was inhibited to a lesser e x t e n t and t h e r e was no effect on

111

sepiapterin reductase from human liver. M H a T P present in some substrate (NH 2 TP) preparations shows the same effect but so far we are not able to say if it is an effect of its oxidized form MTP or M H 2 T P itself. TABLE 1 EFFECT OF SOME PTERINS ON ACTIVITY OF 6-PYRUVOYL TETRAHYDROPTERIN SYNTHASE AND SEPIAPTERIN REDUCTASE

Compound

Concentration jjmol/l

none

-

Enzyme activity (%) PPH4S PPH4S SR human salmon human 100

100

100

45

100

L-Monapterin

24

5

L-H 2 -Monapterin

24

100

-

-

L-H4-Monapterin

24

100

-

-

D-Monapterin

24

100

-

L-MH 2 TP

64

79

-

PPH^S-activity SR-activity

100

-

: pmol BfU/imin· mg protein) : pmol S red./(min-mg protein)

Incubations of L-monapterin with mouse myeloma cell lines X63 Ag8 which are not able to produce tetrahydrobiopterin and in which dihydropterin is the main metabolite showed significant decrease in intracellular pterin production (Tab. 2). There was no effect when incubations were performed with D-monapterin. These experiments indicated that in vivo the inhibition of PFH4S might induce a deficiency of pterins.

112

TABLE 2 I N H I B I T I O N O F P T E R I N B I O S Y N T H E S I S IN M O U S E M Y E L O M A C E L L L I N E S BY L - M O N A P T E R I N

Cell line X63 Ag8 incubated with

N u m b e r of cells

Ρ Μ p m o l / 1 0 m i o cells

IMDM

17 m i o

25

IMDM + ImM D - M o n a p t e r i n

15 m i o

27

299

IMDM + ImM L - M o n a p t e r i n

12 m i o

10

248

Medium

: IMDM + 10% F C S + a n t i b i o t i c s + m e r c a p t o e t h a n o l

Incubation

: 6 d a y s at 3 7 * C and 5.5% C 0 2

To investigate further the effect of monapterin on t e t r a h y d r o b i o p t e r i n b i o s y n t h e s i s in v i v o a g r o u p of 5 mice were treated for three days with monapterin. 1.15 mg of m o n a p t e r i n in 0.1 Μ NaCl, pH 7.3, w e r e i n j e c t e d into a C57 BL/6 male mouse for t h r e e days, and 12-h u r i n e s were collected. A f t e r t h e m o n a p t e r i n d o s e t h e m i c e w e r e limp and t h e i r c o a t w a s s t a n d i n g up. O n e m o u s e d i e d on t h e f o u r t h day and its blood, liver and brain were collected. The serum p h e n y l a l a n i n e levels r e m a i n e d u n c h a n g e d u p o n t r e a t m e n t with m o n a p t e r i n . U r i n a r y b i o p t e r i n d e c r e a s e d r a p i d l y to a b o u t 25% of t h e initial value, and n e o p t e r i n w h i c h n o r m a l l y c o u l d not be d e t e c t e d in m o u s e was e x c r e t e d 12 h o u r s after the first i n j e c t i o n of m o n a p t e r i n (Fig. 3). This e x p e r i m e n t s h o w e d for t h e f i r s t t i m e t h a t m o n a p t e r i n can lower u r i n a r y e x c r e t i o n of b i o p t e r i n in mice. Inhibition of PPH Ζ •t

«λ aj ι c >.-σ — "" Ίο φ ·· c ο ω Ο Φ 3 _ ω I ,— J3 I— Q-U- -Q Ο. O C —' « I 1/1 c

C O et

ο ζ 3: ·-• ι— ο: «r ι X Ο. s ι [3D

ϊ Ο

φ

paz L LXi|do^ [

Ό ' -

I

χ: ο

C ·> -L ϊ1- +-* Φ >1 Sφ ·— Ο- φ C Ιο Φ -Ο U LLJ ε • TD I— ΙΛ φ >- σι Ο C ο^-Οί . Ο Ε 3: -α > LlJ _Q Ο Οί ·

ßui

Od ΓΟ >3:

ιΛ c

J. φ ο < ο >- TD Li- Ο U. Ο

>Ο ο C O ο Σ : Ό Ο ο α·.

/ßd

• ο -σ.

ο Ε -Μ Ο S- SU- + φ -> ΗLiJ (Ο >· >ι ΟΊ^.1— Ο _1 >> I 3: .-• ε οο α- ο ς: ζ +-> >- Ο 84% and 98%,

of these p t e r i n s

in t h e i r

BIOPTERIN

reduced

forms

respectively).

As e v i d e n t from T a b l e 4 , more than 50% o f the n e o p t e r i n , p t e r i n and b i o p t e r i n fluids

exists

present

detection,

the one hand not d e t e c t a b l e

tissue.

moiety p r e s e n t blastic

10. T h i s

acidic conditions biopterin

is

These reduced p t e r i n s

and chemical

The f r e q u e n t l y

concerns

conversion

used

and of i t s

on

thereeven CSF

biopterin

(e.g.

1ympho in

the

are very s e n s i t i v e even under

to

less

temperatures.

(11,12,13,14,15,16,17,18)

"extraction"

of

reduced forms from u η ο χ i d i ζ e d b i o l o g i c a l

samples by a u t o c l a ν i ng ( a p p r o x . , bioassay with C r i t h i d i a of reduced p t e r i n s .

is

the

known to e x i s t

reactions,

and at i n c r e a s e d

HPLC

in part

in p a r t i c u l a r

i n C S F , u r i n e or i n t r a c e l l u l a r l y

tetrahydro-form. oxidation

detection markedly,

biopterins)

Using

samples and

On the o t h e r h a n d , most o f the

and l y m p h o c y t i c

biological

the reduced p o r t i o n

in n o n - o x i d i z e d

the a n a l y t i c a l

by a f a c t o r of about and t o n s i l

and i n

i n the d i h y d r o - or t e t r a h y d r o - f o r m .

methods w i t h f l u o r e s c e n c e fore decreases

intracellularly

mona-

10 min at

fasciculata

leads

12 0°C) p r i o r to some

(Note t h a t some p t e r i n s

to

conversion

o t h e r than

bio-

132 pterin/reduced

biopterins

pterin, sepiapterin, thro-neopteriη

(34)

in

). The m e c h a n i s m

tions ported

buffer

in d e t a i l

18%

(!) of o r i g i n a l means

after

less

above

the

as

levels

(after

detection

de-

- 18) at

reac-

forms)

temperatures.

et

in As

re-

biopterin

moiety

pH 4 . 5 , a n d

only

was

converted

autoclaving

whole

limit.

less

than

of l y m p h o b l a s t i c

blood)

10 - 20%

lymphocytic

of u n o x i d i z e d w h i t e

would

In t h i s

or

be at or

connection can h a r d l y

cells

somewhat

intracellular be

demonstrated

significant.

Occurence

of m o n a p t e r i n

We h a v e a l r e a d y neopterin) blood from We

the

5 - 6 ml

of

and

forms.

that even

alterations

reactions

conversion

( 2 1 ) , 55% of the (11

J}-ery-

and Zeitler

a m o u n t of t e t r a h y d r o b i o p t e r i n

stable

than

(20)

on the

increased

autoclaving

tetrahydrobiopterin from

et a l .

schemes

and at

by M i l s t i e n

destroyed

to o x i d i z e d ,

some

by P f l e i d e r e r

forms; biopterin/reduced

systems

was

This

Fukushima

demonstrated

(neopterin/reduced

different

L.-erythro-neo-

of a u t o x i d a t i o n

was e l u c i d a t e d

(19), likewise

al . (4) h a v e

Crithidia-active:

6-hydroxymethyl pteriη, monapterin,

of t e t r a h y d r o b i o p t e r i n scribed

are

and

reported its

know

diseases

nothing

monapterin

about

in h u m a n

in h u m a n shima thetic

in rat

urine

in p l a s m a

in h e a l t h y the

liver

by F u k u s h i m a

and

patients

of

was

. As e a r l y

proposed

that

the

pathway

from

GTP to t e t r a h y d r o b i o p t e r i n

(28).

and neopterin

Twelve years

concentrations

tetrahydrobiopterin

were

deficiency

reported

in n e w b o r n s

the

described

as

1970,

in

of the

are

urinary for

of

first

metabolites

increased

role

phenylalanine

some

(29).

Fuku-

biosyn-

dihydroneo-

( d i h y d r o m o n a p t e r i n ) , and

later,

suffering

et al . r e p o r t e d

as a c o f a c t o r

intermediary

peripheral

and b i o c h e m i c a l

cells.Iwai

et a l .

of the

(threo-

.

(27). M o n a p t e r i n

pterin, threo-dihydroneopteriη pterin

subjects

biosynthesis

be a c t i v e

of m o n a p t e r i n

and cells

(2-4, 9, 22-26)

and animal

reduced monapterinto hydroxylase

on the o c c u r e n c e

levels

and b o n e m a r r o w various

( t h r e o - n e o p t e r i η ).

sepia-

monapterin cases

of

133 In o u r urine

study

on n o r m a l

(see T a b l e s

and monapterin compartments found

human

2 - 4 ) , the

as well

is e v i d e n t .

Analogous

findings

were

malignant

diseases

(tumor,

monapterin

simultaneous

concentrations

were accompanied

than

obtained

in m o s t

levels

cells,

as t h e i r n e a r l y

investigated

in h i g h e r

blood

before

occurrence

constant

in p a t i e n t s

by a n a l o g o u s

and during

pancreas

and

cyte

fraction,

The f a c t

that

threo-isomer systems Table

the

ago

EPIMERASE

which

results

moiety ative

type

neopterin

(L-) would

related

intermediates

(substrate,

rather

in the

now t h a t the reaction

group

a hydroxymethyl

The occurrence a n d e f f e c t tetrahydrobiopteriη

and

its

the

cell

and

(see

reported

cell

of

systems

phosphorylated

neopterin The

of

deriv-

epi-

L-monapterin

occuring

euglena-

steps

cascade

at

the

between

di-

L^-tetrahydrobiopterin:

phosphate-eliminating triphosphate)

reaction

results

at C - 3 ' . H o w e v e r ,

group

leuco-

reaction

reduction

biosynthesis and

beta-

reaction

occurs

before

in the

formation

(30);

phosphate-eliminating

of a m e t h y l

animal

formation by

the

caused

-as

triphosphate).

than

the

impressive

an e n z y m i c

the

in

peripheral

f o r us

to the n a t u r a l l y

dihydroneopteriη

reduction

is

of a r e d u c e d

explain

neopterin

cytostatics

in all

f r o m an a p p r o p r i a t e

triphosphate

It is a s s u m e d

b) The

formation

from ^ - n e o p t e r i n

hydro

obvious

D-dihydroneopterin

reaction

of

identical

to s u g g e s t

with

neopterin

investigated

it was q u i t e

from

(26).

of b o t h

nearly

in

affects

in the

urine

portion

the

always

hyperglykemia,

in m o n a p t e r i n - f o r m i n g

in the

level

the

fluids/urine

is c l o s e l y

pterin)

and

was

(22, 24)-

(probably

(which

any

reduced

of m o n a p t e r i n

merization

a)

plasma

4). T h e r e f o r e ,

induces

in b o t h p t e r i n s

monapterin

and body

some y e a r s the

the

in

suffering

treatment

cells

decrease

was

neopterin

alterations

Proglicem, which

a significant

of

ratio

alterations

(22-25).

human

and

monapterin.

and/or antibiotics in the

fluids

Neopterin

leukemia):

cases

body

( - C H ^ O H ) at

monapterin

has

C-3 .

of d i h y d r o m o n a p t e r i η

biosynthesis

still

1

in v i t r o

and

triphosphate in v i v o

is

on

dis-

134

cussed

in t h i s

volume e l s e w h e r e

There have been many r e p o r t s (or i t s

reduced f o r m s )

the p o t e n t i a l roles

(35).

on the o c c u r r e n c e of

in several

importance

of t h i s

microorganisms.

investigated,

Methylococcus hol

is

dehydrogenase

the c y c l i c

(32).

In S e r r a t i a

m e t h y l p t e r i n were i d e n t i f i e d merization

triphosphate pterin

(33)

epimerase ),

i n E. c o l i

of J_-difor

alco-

p h o s p h a t e and 6 - h y d r o x y from GTP

(27).

r e p o r t e d an enzyme-dependent to

epi-

dihydromona-

( ' JD-ery t h r o - d i h y d r o n e o p t e r i η

the b i o c h e m i c a l

remained

in

i n d i c a j ) - e r y t h r o - n e o p t e r i η,

as p r o d u c t s

i n E. c o l i 1

( 3 1 ) , and

as a c o - f a c t o r

of j)-di h y d r o n e o p t e r i η t r i p h o s p h a t e

pterin triphosphate

micro-

i n the

monophosphoester

t h o u g h t to f u n c t i o n

Brown et a l .

two o f the

to t y r o s i n e

L^-mona p t e r i η , J.-mona p t e r i η 2 ' , 3 ' - c y c l i c Although

metabolic

as a c o - f a c t o r

of p h e n y l a l a n i n e

capsulatus

hydromonapteriη

indicates

i n Comamonas s p . , a reduced form of

m o n a p t e r i n was d e c r i b e d to f u n c t i o n enzymic h y d r o x y l a t i o n

This

p t e r i n , although

f o r m o n a p t e r i n have been assumed i n o n l y

organisms

monapterin

f u n c t i o n of mona-

unknown.

Conclusions ( 1 ) Human bone marrow p r e s e n t e d h i g h e r concentrations (2)

In peripheral

than p e r i p h e r a l blood c e l l s ,

found to be a p p r o x i m a t e l y (3)

Not o n l y b i o p t e r i n , (e.g.,

(4)

CSF, t o n s i l

within

nearly

identical

ii)

nearly

constant

b i o p t e r i n was

the same

range.

in t h e i r

reduced

were

forms

lymphocytes).

As an i n t e r p r e t a t i o n enzymic

cells.

investigated,

NEOPTERIN and MONAPTERIN

i)

pteridine

n e o p t e r i n and m o n a p t e r i n

m a i n l y

In each of the compartments threo-isomers

blood

intracellular

but a l s o

found to be p r e s e n t

intracellular

portions ratios

of t h e i r

the

presented reduced

forms,

(neopterin/monapteriη;

of these f i n d i n g s

N/M).

we s u g g e s t an

r e a c t i o n of the EPIMERASE type between

neopterin triphosphate

erythro-

and L - d i h y d r o m o n a p t e r i η

D-dihydrotriphosphate.

135

References 1. Huber, Ch., J.R. Batchelor, D. Fuchs, Α. Hausen, A. Lang, D. Niederwieser, G. Reibnegger, P. Swetly, J. Troppmair. H. Wächter. 1984. J. Exp. Med. J60, 310. 2. Andondonskaja-Renz, B., H.-J. Z e i t l e r . 1986. In: Chemistry and Biology of Pteridines (B.A. Cooper, V.M. Whitehead, eds.). De Gruyter, Berlin-New York, p. 431. 3. Zeitler, H.-J., B. Andondonskaja-Renz. 1987. In: Biochemical and C l i n i cal Aspects of Pteridines (W. Pfleiderer, J.A. B l a i r , H. Wächter, eds.) De Gruyter, Berlin-New York. Vol 5 (in press). 4. Z e i t l e r , H.-J., B. Andondonskaja-Renz. 1986. In: Methods in Enzymology (S.P. Colowick, N.O. Kaplan, F. Chytil, D. McCormick, eds.). Academic Press, Orlando, USA. Vol. 122, p. 273. 5. Pertoft, H., A. Johnsson, Wärmegärd, Β., R. S e i j e l i d . 1980. J. Immunol. Methods 33, 221. 6. Gutierrez, C., R.R. Bernabe, J. Vega, M. Kreisler. 1979. J. Immunol. Methods £9, 57. 7. Ulmer, A.J., H.D. Flad. 1979. J. Immunol. Methods 30, 1. 8. Gmelig-Meyling, F., Τ.Α. Waldmann. 1980. J.Immunol. Methods 33., 1. 9. Zeitler, H.-J., B. Andondonskaja-Renz, G. Küther, A. Struppler. 1986. In: Chemistry and Biology of Pteridines (B.A. Cooper, V.M. Whitehead, eds.). De Gruyter, Berlin-New York, p. 251. 10. Fukushima, T., J.C. Nixon. 1980. Anal. Biochem. H)2,

176.

11. Ziegler, I . 1985. In: Biochemical and Clinical Aspects of Pteridines (H. Wächter, H.Ch. Curtius, W. Pfleiderer, eds.). De Gruyter, BerlinNew York, Vol. 4, p. 347. 12. Fink, M., U. Jehn, W. Wilmanns, I . Ziegler. 1982. In: Biochemical and Clinical Aspects of Pteridines (H. Wächter, H.Ch. Curtius, W. Pfleiderer, eds.). De Gruyter, Berlin-New York, Vol. 1, p. 241. 13. Ziegler, I . , M. Fink, W. Wilmanns. 1982.

Blut 44,

231.

14. Ziegler, I . , H.J. Kolb, U. Bodenberger, W. Wilmanns. 1982. Blut 44, 261. 15. Fink, M., U. Jehn, W. Wilmanns, H. Rokos, I . Ziegler. 1983. In: Biochemical and Clinical Aspects of Pteridines (H.Ch. Curtius, W. Pfleiderer, H. Wächter, eds.). De Gruyter, Berlin-New York, Vol. 2, p. 223. 16. Fink, Μ., I . Ziegler, H. Rokos. 1985. In: Biochemical and Clinical Aspects of Pteridines (H. Wächter, H.Ch. Curtius, W. Pfleiderer, eds.). De Gruyter, Berlin-New York, Vol. 4, p. 387. 17. Fink, Μ., I . Ziegler, H.J. Kolb, K. Demtröder, V. Nüssler. 1987. 1th Workshop on Unconjugated Pterins and Related Biogenic Amins. February 28 - March 7, Flims, Switzerland. 18. Fink, Μ., V. Nüssler, Κ. Demtröder, Ε. Höniges, I . Ziegler, Μ. Goldberg. 1987. 6th Winter Workshop on Biochemical and Clinical Aspects of Pteridines. February 14-21, St.Christoph/Arlberg, Austria. (See also this volume).

136

19. Pfleiderer, W.. 1975. In: Chemistry and Biology of Pteridines (W. P f l e i derer, ed.). De Gruyter, Berlin-New York, p. 941. 20. Fukushima, Τ., J.C. Nixon. 1979. In: Chemistry and Biology of Pteridines (R.L. K i s l i u k , G.M. Brown, eds.). Elsevier, North Holland, p. 31. 21. M i l s t i e n , Sh.. 1983. In: Biochemical and Clinical Aspects of Pteridines (H.Ch. Curtius, W. Pfleiderer, H. Wächter, eds.). De Gruyter, Berlin-New York, Vol. 2, p. 65. 22. Andondonskaja-Renz, B., H.-J. Zeitler. 1984. In: Biochemical and C l i n i cal Aspects of Pteridines (W. Pfleiderer, H. Wächter, H.Ch. Curtius, eds.). De Gruyter, Berlin-New York, p. 295, Vol. 3. 23. Andondonskaja-Renz, B., H.-J. Zeitler. 1985. In: Biochemical and C l i n i cal Aspects of Pteridines (H. Wächter, H.Ch. Curtius, W. Pfleiderer, eds.). De Gruyter, Berlin-New York, Vol. 4, p. 559. 24. Z e i t l e r , H.-J., B. Andondonskaja-Renz. 1984. In: Biochemical and C l i n i cal Aspects of Pteridines (W. Pfleiderer, H. Wächter, H.Ch. Curtius, eds.). De Gruyter, Berlin-New York, Vol. 3, p. 313. 25. Z e i t l e r , H.-J., B. Andondonskaja-Renz. 1986. In: Cancer Detection and Prevention (The International Society for Preventive Oncology) (H.E. Nieburgs, ed.). A.R. L i s s , Inc., New York, (in press). 26. Z e i t l e r , H.-J., B. Andondonskaja-Renz. 1985.

(Unpublished data).

27. Iwai, Κ., M. Kobashi. 1975. In: Chemistry and Biology of Pteridines (W. Pfleiderer, ed.). De Gruyter, Berlin-New York, p. 341. 28.

Fukushima, T.. 1970. Arch. Biochem. Biophys. 139, 361.

29. Niederwieser, Α., A. Matasovic, W. Staudenmann, Μ. Wang, H.Ch. Curtius. 1982. In: Biochemical and Clinical Aspects of Pteridines (H. Wächter, H.Ch. Curtius, W. Pfleiderer, eds.). De Gruyter, Berlin-New York, Vol. 1, p. 293. 30. S. Ghisla. 1987. 1th Workshop on Unconjugated Pterins and Related Biogenic Amines. February 28-March 7, Flims, Switzerland. (See this volume). 31. Guroff, G., C.A. Rhoads. 1969. J. Biol. Chem. 244, 142. 32. Urushibara, T., H.S. Forrest, D.S. Hoare, R.N. Petel. Biochem. J. 125, 141.

1971.

33. Brown, G.M., J. Yim, Y. Suzuki, M.C. Heine, F. Foor. 1975. In: Chemistry and Biology of Pteridines (W. Pfleiderer, ed.). De Gruyter, Berlin-New York, p. 219. 34. Nixon, J.C.. 1985. In: Folates and Pterins (R.L. Blakley, St.J. Benkovic, eds.). J. Wiley&Sons, Inc., New York, Vol. 2 (Chemistry and Biochemistry of Pterins), p. 1. 35. Blau, Ν., P. Steinerstauch, G. Schoedon, R. Redweik, A. Niederwieser. 1987. 1th Workshop on Unconjugated Pterins and Related Biogenic Amines. February 28 - March 7, Flims, Switzerland. (See this volume).

MODIFIED

PTERIPINE

LINES

DURING

Seidl 1 ,

J. I.

1

AND

für

für

Ileinschad1 ' ·

Ch.

Experimentelle

D-8000

Institut

Mainz,

TRANSFORMED

CELL

SHOCK

Strahlen

strafte 61, 2

IN

and

1

Institut

schaft

HEAT

Przyby 1 sk i 2 ,

M.

Ziegler

SYNTHESIS

für

und

2,

Gesell-

Landwehr-

FRG

Organische 18-20,

der

Unweltforschung,

München

Becherweg

HSmatologie

Chemie

D-6500

der

Mainz,

Universität

FRG

Introduction

A

pteridine

found

to

lignant a

binding

accumulate diseases

chromophoric

guanidine that

the

nate

by

cell

from

lines

over,

we

and have

to

sponse set

of

certain *New

is

of

its

cells

proteins stages

address:

be

of

cell

to

normal folate

found

shock

In

the

pteridine

cells

this

is

is

differentiation Mannheim

in

trans-

(5)

origi-

study

we

cleavage.

have More-

characterize in

heat

normally

Boehringer

this

by 6 Μ

indicated

pteridines

biogenesis.

heat

which

with

lines.

In

by

ma-

characterized detached

pteridine.

chemically

induction

was

with

analysis a

that

and

(AGPH)

patients

that

postulated

its

to

is

show

typically

elucidate

studied

was

claimed

begun

which

AGPH could

cleavage.

this

have

pteridine

we

(2,3,4)

folate

reinvestigated

of

. Spectral

transformed been

glycoprotein

blood

group

experiments

earlier

formed

which

hydrochlorid

synthesized had

the

( 1 ) . This

group

chromophoric

following It

ofi-acid in

malignant

the

cells

Additional1y, shock, known

become (for

since

to

induce

active review

GmbH,

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

Werk

we rea

during see

6).

Penzberg

138

Material

and

methods

H e L a , H T - 2 9 a n d 2 3 9 c e l l s w e r e c u l t u r e d in M E M w i t h Ι Ο * N B S a n d 1 0 * F C S , r e s p e c t i v e l y . R e h , HL 6 0 a n d T 4 7 D w e r e g r o w n in R P M I w i t h 1 0 * F C S . To study f o l a t e m e t a b o l i s m , medium folate was rep l a c e d by 1 4 C - f o l a t e (Amersham; final concentration 1,5μΜ, specific activity lpCi/18 nmol). H e a t s h o c k w a s p e r f o r m e d by c u l t i v a t i n g t h e c e l l s a n d b a c t e r i a at 4 3 · C f o r 4 h o u r s . R e v e r s e p h a s e H P L C d e t e r m i n a t i o n of p t e r i d i n e s a f t e r acidic iodine oxidation is described in (7). The amount of pterin X was calculated by assuming a f l u o r e s c e n c e i n t e n s i t y of b i o p t e r i n . Fast atom bombardment (FAB) mass spectra were obtained with a modified Finnigen MAT-312 spectrometer, equipped with a h i g h f i e l d laminated magnet system and extended analyzer. A thermal Cesium primary ion source (AMD I n t e r t r a , B e e k e l n / F R G ) w a s e m p l o y e d and operated at ca. 1400 mA and 4.5 kV (relative to s o u r c e p o t e n t i a l ) . A l t e r n a t e a c q u i s i t i o n of p o s i t i v e and negative ion spectra was carried out using a post-acceleration / ion conversion dynode system (AMP) placed in—line before the 17-stage electron m u l t i p l i e r , e m p l o y i n g a d y n o d e v o l t a g e of + 8 kV f o r negative conversion. Lyophilized s a m p l e s of t h e c h r o m a t o g r a p h i c a l l y isol a t e d p t e r i n X of a p p r o x . 10 μ β w e r e d i s s o l v e d in c a . 5 μ ΐ m e t h a n o l : IM H C l ( 5 : 1 ) , a n d l - μ ΐ a l i q u o t s w e r e a d m i x e d t o 0 . 5 μ ΐ of g l y c e r o l or t e t r a e t h y l e n e g l y c o l (TEG) as matrix substance. After brief treatment in an u l t r a s o n i c b a t h , t h e r e s u l t i n g , n e a r l y homogenous s o l u t i o n w a s a p p l i e d on a 2 m m 2 s t a i n l e s s s t e e l F A B t a r g e t at t h e t i p of a s e l f - c o n s t r u c t e d , thermostated direct insertion probe. Samples were introduced in t h e i o n s o u r c e a n d s p e c t r a a c q u i r e d at 17 s e c / d e c a d e by t h e F i n n i g e n S S - 2 0 0 d a t a s y s t e m ; a r e s o l u t i o n of ca. 2 . 0 0 0 (10*) was employed. Results

Origin Fig.L

and

discussion

of A G P H c h r o m o p h o r e demonstrates that the

chromophore

of

AGPH

is

139

PTERIN Χ

ο a

NEQPTERIN

PIE-

Τ

4

-τ-

—Γ"

6

8

—r- r—r 10

12 min

Fig.1 HPLC separation (1% acetonitrile, 7% methanol, 3mM phosphoric acid) of the chromophoric group of AGP (lower lane) and of HeLa pteridines (upper lane) 6- c a r b o x y p t e r i n [1

p t e r i n - 6 - carboxaldehyde

18 pmol folic

6000 e

CL

70 pmol acid 225 pmol ι

4000

°

2000

Timefinin]

Fig.2 HPLC separation (10% methanol) of commercially obtained C-rfolate absorption at 280nm, - - - fluorescence (exc. 350nm, em. 450nm), dotted area: radioactivity

140

identical

with

cells.

all

In

methods) far.

this

Folate

14

section

of

below).

had

originated

of

gave

*C-folate

freshly

the

be

the

at

that not to

predominated present

1

by

only

in

with

alone.

HeLa in

with

3

HPLC

the

already

whether

and

the

had

and

purified

by 24 or

of

unconjugated

used

sam-

unpurified

the

incubation we

conclude

pteridines

folate

degradation

acti-

purified

cells,

may

the

cells.

unconjugated

medium

folate

HeLa

the

To

cells/ml)

Therefore,

by

6-

(fig.2). for

of the

extended

days.

the

40*

one

as

incubated

period

(2,3,4,5)

of

was

of

that

present

(3.6x10*

without

cleavage of

sample

fraction

of

8

they

separation

demonstrated

was

this

or

to

about

if

obtainable

the

impurities

enzymatic

showed

(see

pteridines

cells

After

maximal

shock

expected,

subsequently

preparations

portion

heat

commercially

Indeed,

irrespective

folate

cells,

by

unconjugated

the

*C-folate

and

folate

HeLa

pteridines

that

impurities

of

in

pterin-6-carboxaldehyde

labelling to

serum to

C

period

both

bute

e.g.,

cleavage.

in

investigators

the due

The

(see

X)

activity

folate

recovered

C-folate a

and

incubated

Earlier over

HeLa

were

induced

radioactivity

37°

pteridines,

14

by

investigated

culture

isolated

notion

HPLC

medium

was

was

present

these

was

cell

preparation.

preparative

ple

(pterin

specific

manufactured

of

eliminate

vity

lines

cleavage X

from

carboxypterin

with

HeLa

The

the

us

already

hours

in

pterin

third

third

produced,

cell

biopterin

C-folate

one

1

and

cleavage

synthesis

may

6

amounts.

examine

This

pteridine the

pteridine

Neopterin

minimal

To

a of

but

was

rather

decomposition.

possibly (for

contri-

review

see

8) .

Induction oxygen HeLa to

of

pterin

X

by

heat

shock

and

by

limited

supply

cells

limited

which oxygen

amounts

of

pterin

tab.l).

Neopterin

had

been

supply X and

as

subjected accumulated

compared

biopterin

to

to at

heat

least

controls

levels

shock

were

or

3-fold (fig.3,

not

rai—

141

|

r

Μ I Pterin X

Ncopterin. 0.07 pniot Bioptcrin 0. Η pmol Pterin X : 0,65 pmol

Ne opterirn 0,34 pmol Biopttrin ; 0,18 pmol Pterin X 3,03 pmol

ι—

F i g . 3 H P L C s e p a r a t i o n of p t e r i d i n e s f r o m H e L a (10 cells) upper lane: control, l o w e r l a n e : r e d u c e d o x y g e n cond i t i o n (2 hours)

•0

120

240

100

Fig.4 U V - s p e c t r u m of p t e r i n X - · - · - pH1, p H 7,

140

Ι·0

DH

«20

13

M0

nm

142

sed

by

stress

phorbol

ester

of

these

both

Heat

pterins

shock-induced

stricted

to

accumulate mally only

conditions. treatment

60

at

least

reported

mally

contrast

an

accumulation

of

pterin

Bacteria,

X

such

is

as

to

not

re-

E.COLI,

also

3-fold

levels

of

pterin

protein).

Levels

of

monapterin,

pteridine

shock-i nduced

micked

in

E.COLI (10),

by

synthesis

incubation

of

sepiapterin

(SEP).

gether

sepiapterin

with

pterin

bination

X.

Maximal

(tab.l).

were

X

(maxi-

only

(MTX). (DHFR)

Temp.

This

Pteridines

controls

at

cells

reached

the

mini-

is

43eC

the

by

X

be

37°C

to

and

is

that

miwith to-

amount

this

induced

pterin

indicates

activity

can

increases

are of

X

cells

the

heat-shock

by

reductase

HeLa of

levels

Both

pterin

further

accumulation

methotrexate

of

the

Transfer

pterin-mediated late

in

raised.

Heat

of

is

causes

(9).

synthesis

eucaryotes.

pmol/mg

This

which

com-

sepia-

inhibited dihydrofo-

essential.

pmol/106cells

+SEP(1)JM)

+SEP(1jjM)

+ΜΤΧ(1μΜ)

+Γ1ΤΧ ( IJJM ) 37° C

Biopterin

0. 4 5

Pterin X

1.94

5. 4 3 3. 0 7

Biopterin

0. 20

Pterin X

4. 77

43° C

Tab.l

It

is

Accumulation (106

cells)

n.d.

= not

known

sepiapterin reductase which (tab.l) However, ted

seem the

6. 9 2

2. 13

0. 12

9. 0 9

4. 6 3

1. 3 2

of pterin with

that

X by incubation

sepiapterin

(SEP)

and

tetrahydrobiopterin successive

DHFR

found to

(11). in

n.d.

of HeLa by heat

of

suggest

may

reduction Increased

the

originate

data

reduction

n.d.

shock;

determined

via

and

were

1. 1 8 0. 74

this

that

sepiapterin

a

arise

of

further

levels

sepiapterin

"salvage

occurs.

from

sepiapterin

biopterin

presence

from

by

pathway".

DHFR-media-

Iodine

oxida-

143

t i o n (at p H 1) of t h i s p r o d u c t y i e l d s p t e r i n X. The product, which results from sepiapterin after incubation with DHFR and NADPH, was identified as dihydrosepiapterin (12). Using the conditions described in (12). a linear progression of dihydros e p i a p t e r i n f o r m a t i o n w a s o b t a i n e d d u r i n g a p e r i o d of 2 0 m i n u t e s . I o d i n e o x i d a t i o n (at p H 1 ) of t h e i n c u b a tion product, dihydrosepiapterin, under acidic conditions yields pterin X. T h e KM f o r sepiapterin in D H F R - m e d i a t ed r e d u c t i o n ( b o v i n e l i v e r ) is 18 f*M a n d t h e Hi for m e t h o t r e x a t e is 2 nil ( f i g . 4 ) . This indic a t e s t h a t t h e a f f i n i t y of s e p i a p t e r i n a s a s u b s t r a t e is c o m p a r a b l e t o t h a t of d i h y d r o f o l a t e (13). From the r e s u l t s described a b o v e we c o n c l u d e that in transformed cell lines and under heat shock conditions a D H F R - d e p e n d e n t r e d u c t i o n of s e p i a p t e r i n occ u r s . It r e m a i n s t o b e d e t e r m i n e d whether resulting dihydrosepiapterin or its o x i d a t i o n product, pterin x, is p r e s e n t in t h e l i v i n g c e l l . T h e h e a t s h o c k — i n duced formation of dihydrosepiapterin appears to branch off the de novo pathway, which results in tetrahydrobiopterin and can be triggered in transformed cell lines by phorbol ester treatment. In c o n t r a r y t o h e a t s h o c k i n d u c e d s y n t h e s i s of p t e r i n X, phorbolester treatment causes a transient accumulat i o n of n e o p t e r i n a n d b i o p t e r i n (9) w h i c h c a n n o t be inhibited by m e t h o t r e x a t e . The key compound in the h e a t s h o c k - i n d u c e d p a t h w a y a n d t h e m e c h a n i s m s of t h e divergence from the de novo pathway remain to be determined.

C h e m i c a l c h a r a c t e r i z a t i o n of p t e r i n X P t e r i n X is h a r d l y t o b e d i s t i n g u i s h e d by H P L C from 2-amino-4-oxo pteridine (pterin). Only with phosphate b u f f e r a s an e l u e n t (15 m M , p H 6 , 4 ) a s h o u l d e r indicates a slightly different retention time. T h e s p e c t r a l d a t a a r e s h o w n in ( f i g . 5 ) . T h e product of alkaline ΚΓΙηΟ* oxidation comigrates with 6-carboxypterin, thus indicating a carbon side chain on p o s i t i o n C6 (14). Initial m o l e c u l a r weight and s t r u c t u r a l data for p t e r i n X w e r e s o u g h t f r o m F A B m a s s s p e c t r a . In t h e s e e x p e r i m e n t s , t h e p r e p a r a t i o n of s u i t a b l e m a t r i x systems

144

Fig.4 L i n e w e a v e r - B u r k p l o t of s e p i a p t e r i n r e d u c t i o n b y D H F R (bovine liver) a n d N A D P H ( 50 m M ) a n d of M T X i n h i b i t i o n

iee-i [M + Η*] a 218 B0-

(β

2β-

ιββ

1SB

200

F i g . 5 F A B m a s s s p e c t r u m of p t e r i n X

25«

300

350

145

was

found

(though tained these

to

not

be

of

entirely)

after or

abundant

protonated

salt

the in

TEG

at

pterin

X.

in

positive

absence

to

m/z

indicating The

of

lack

of

labile,

matrix

any

structure,

vation

more

but

detailed

a

of

of

chain

precluded

de-

sodium spectra ions

217

the

a to

for

fragment

spectra

structural

most

218,

ion

weight

side

At

attributable

abundant

ion

functional

m/z

background

(1C1)

more

ob-

either

yielded at

amounts

and

were

with

negative

molecular

negative

piapterin of

the

252/254

a

and

MH +

ion

nearly

methanol/HCl.

spectra

considerable

sample;

addition ion

(M+Cl) - ,

of

in

consistently

molecular

isolated

in

prominent

the

matrix

presence

the

showed,

as

ion

and

solutions

protonation

positive

glycerol

importance,

homogenous

enforced

conditions,

spite

crucial

ions

suggests

the

from

se-

the

direct

data,

deri-

from

these

spectra.

References 1.

Ziegler,

I.,

Research 2.

Fukushima, 249,

3.

K.

42_, T.,

R.,

Conklin,

B.

Clark,

Halpern,

D.

Hardy,

B.C.

74^

Stea,

Β.,

B.C.,

Halpern,

P.S.

Clynes,

M.M.,

Letters

10.,

Lanks,

7.

Ziegler,

8.

Scott, (R-L. John

T.

Shiota.

K.W.

Cancer

1974.

J.Biol.Chem.

B.

Stea,

A.

Ashe,

A.

L.

Smith.

Dunlap,

Sperling, 1977.

J.A.

Proc.Natl.

587.

Backlund, 38,

and

P.B.

Halpern,

Berkey, R.A.

A.K.

Smith.

Cho,

1978.

2378.

C.

O'Neill.

1980.

Cancer

133. 1986.

Exp.

1985.

J. Ce 11. Β i ochem.

28.,

J.M.

1984.

In:

Pteridines

Blakley J.,

Rembold, Z.

1982.

I.

Wiley

Seidl,

H.

& M.

and

S.J.

Sons.

p.

Cell

H.

Physiol.

G.

Chem.

and

Benkovic, and

Heinz. 352,

165,

1. 197.

ed.)

307.

Borchert,

and

Res.

Folates

Biochem.Biophys.Res.Com. 10.

Fink.

Halpern,

R.M.

Research

6.

9.

and

Halpern,

Cancer 5.

M.

4445.

Acad.Sei.USA 4.

Maier,

1567.

I.

141,

Ziegler. 494.

1971.

1271.

1986.

Hoppe-Seyler~s

Κ

146

11. N i c h o l , C . A . , C . L . Lee, Μ . P . E d e l s t e i n , J . Y . Chao., D.S. Duch. 1983. Proc.Natl.Acad.Sei.USA 80, 15 46. 12. S m i t h , G . K . , and C . A . N i c h o l . 1 9 8 6 . J . B i o l . C h e m . 2 61, 27 25. 13. B l a k l e y , R . L . 1 9 8 4 . In: F o l a t e s and P t e r i d i n e s (R.C. B l a k l e y and S . J . B e n k o v i c , e d . ) , J o h n W i l e y & S o n s . p. 1 9 1 . 14. F o r r e s t , H . S . , H . K . M i t c h e l l . 1 9 5 5 . J . A m e r . C h e n . S o c . , 77, 4 8 6 5 .

Acknowledgement He t h a n k P r o f . D r . A. B a c h e r for m o s t h e l p f u l d i s c u s s i o n s and s u g g e s t i o n s . This research was supported by the Deutsche Forschungsgemeinschaft and by the Bundesministerium für F o r s c h u n g und T e c h n o l o g i e .

SECTION C: PTERINS IN IMMUNOLOGY

DISEASE ASSOCIATED ALTERATIONS OF PTERIN BIOSYNIHESIS: ENHANCEMENT IN INFLAMMATORY DISEASE, HEMOPOIETIC REGENERATION AND ENDOTOXINEMIA

Ch.Huber, M.Herold, J.Trcppmair. Div.Clinical Iimunobiolcy, Dept.Internal Medicine, University of Innsbruck, A-6020

Innsbruck, Austria

H.Rckos Henning Berlin GesiribH Berlin, D-1000 German, Federal Republic

Introduction

Disease associated abnormalities of p t e r i n biosynthesis were f i r s t observed in children with inborn metabolic e r r o r . Atypical phenylketonur i a (PK) i s character!zed by deficiency t o generate appropriate l e v e l s of tetrahydrobiqpterin (BH4) and c l i n i c a l l y presents with CNS dysfunct i o n ( 1 ) . Subsequently, increased l e v e l s of pterins in body fluids were observed in context with disease s t a t e s not a f f e c t i n g brain function. Elevated l e v e l s of necpterin were detected in various inflarmnatory conditions ( 2 ) , during hemopoietic regeneration (3) and in shock p a t i e n t s ( 4 ) . In t h i s a r t i c l e , we suimarize previous and recent data elucidating the pathomechanism(s) underlying increased p t e r i n synthesis in the latter states.

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

150

1. Enhancement of Pterin Biosynthesis During Inflaimnatory Disease Evidence for enhanced pterin biosynthesis during inflarrmatory states i s nainly derived from the observation of increased neqpterin levels in conditions such as viral and bacterial infection, autoiimune disease or allograft rejection. This topic has been recently reviewed (2). In vitro experiments represented the f i r s t clue to unravel the mechanisms underlying this phenomenon. We f i r s t observed that antigenic stinulaticn of a mixture of T-lynphocytes and monocyt es /macrophages leads t o release of iressive amounts of necpterin into culture fluids ( 5 ) . Further studies using highly purified suspensions of T-cells and monocyt es/macrcphages revealed that the macrophage produces necpterin when challenged with T-cell supematants (6). Neutralization experiments and stinulation with various recombinant lynphckines finally enabled t o characterize interferon-garrrra (IFN-garrma) as the active principle contained in these supematants (6). Subsequently, we and others have in detail studied the capacity of various cytokines released frcm inflammatory mononuclear c e l l s t o enhance pterin biosynthesis ( 6 , 1 2 ) . Results are demonstrated in Figure 1. As shown, T-lynphocytes can not be induced fcy any of the cytokines tested. Macrophages cn the other hand are only stimulated with IEN-alpha and IFN-gairma. Peripheral blood mononuclear c e l l s , which are predominantly composed of T - c e l l s and monocytes, are induced t o release necpterin when exposed t o IFN-alpha, IBH-ganna and IL-2. Neutralization experiments with anti-IFN antibodies indicated that the l a t t e r stinulation is indirect and mediated via induction cf interferon-gaima fcy IL-2.

151

Figure 1: Cytokine Specificity and Cellular Cooperations Required for Enhanced Pterin Biosynthesis in Mononuclear Blood Cells

IL·-.! l O n g / H l

ä

Τ + Ho

X I.- i! l O n s / t t l ΧΝF-χOne/Mi I N F - b lOiisi/Ml 1 FN- Ά.

X Ort t f / M

1

Ϊ F N - 3 X Ona/MX GM-CSF lOrig/M 1 i-i^-i-La, ί, I t ) I t I I I I till Mill I 1 I I ,1 Ο ΙΟ Η ΙΟ Ν ΙΟ Ο Η Ν Neoif>te«>in < NMOL/L. P o l t α U a l u e s ) Further experiments, previously published and in detail presented by G.Schoedcn et a l . in this issue aimed t o elucidate the biochemical basis of the phenomenon of IFN-induced enhancement of pterin biosynthes i s (7). A systematic analysis of IFN-induced changes of guanosintriphosphate (GTP), GTP-cyclohydrolase I (GTP-CH I ) , neopterin, biopterin and pterin levels in T-lynphocytes or rronocytes/macrophages gained the following results: IFNs increase substrate concentrations and activate the key-enzyme GTP- CH I in both Τ-eel Is and rronocyt es /macrophages. Macrophages, in contrast t o T-cells are not capable t o further convert dehydronecpterintriphosphate t o BH4. Ihis then leads t o excretion of

152

large anoints of dehydronecpteriη and necpterin into cultur supernatants (7). In conclusion, these in vitro data indicated that both, innune and non-irmune IENs ty selectively activating early steps of pterin biosynthesis in cells of the ncnocyte/macrcphage series lead to increased excretion of necpterin and dehydronecpteriη which is not associated ty enhanced biosynthesis of bicpterin.

Further evidence that I FN mediated enhancement of pterin biosynthesis in certain hemopoietic cells might represent the main cause cf elevated necpterin levels seen during inflammatory disease states are derived frcm in vi\o studies. First, diseases known to be accompanied ty elevated necpterin levels also have detectable levels of circulating IFN (far review see ref.2). Secondly, the view of an intimate relationship between production of endogenous IENs and induction of pterin biosynthesis is further supported ty individuell follow up studies. Exanples for concordant changes of endogenous IEN-levels and necpterin serum concentrations in a patient with cytomegalovirus disease and a patient rejecting his allograft are shown in Figure 2 a and b. Details of such clinical correlation studies were previously published (8,9).

Third, in vivo application of both, reconbinant IEtJ-alpha and reconbinant IFN-gartiTR induce significant elevation of necpterin levels (10,11). In vivo induction of IEN-mediated pterin biosynthesis, as measured ty assessment of necpterin levels is clearly dose dependent (Fig.3). In conclusion, these results prove that activated cells of the monocyte/macrophage series exhibite enhanced pterin biosynthesis. Pterin metabolism in these activated cells is unique because it selectively involves early steps of this pathway and leads to increased production of necpterin without generation of bicpterin. IFNs play a central role ty activating the key enzyme GTP-CH I. These in vitro data and the in vivo studies with recorrbinant interferons indicate that elevated neopterin levels during inflannatory disease reflect the extend of IFNinduced activation of the monocyte/macrophage system.

153

Figure 2: Correlation of Serum Necpterin With IETJ-Ganma Levels in a Patient With CMV-Disease (A) arid a Patient With Acute Rejection (AR) of His Renal Allograft (B)

Ii er· um Hoov'toi'ln < ΜΗΟΙ./Ήαΐι Cr>9ft) or Ζ FH (U/lOOHt) lOOO Nooi>to»>i n 0 00

I FN-Ga>Mi*

η oo

voo GOO aoo

>ίlm^Λr·»β^OWNlt0i>in C ΚΜΟΙ-,'ΜΟΙ, ϋ»οα) ο*· I FH (U/IOOML) 'JOΟ r HooY>feov*ln —β— IFM-aannn 7 0 0

600

BOO

AOO

DAUS

154

Fig. 3. Dose Dependent Enhancement of IETSI-mediated Pterin Biosynthesis

IFN-Gftnnii

F r o n !

Μ Aulltzlcu

a t

Do««

αϊ

(NIarDtfpann)

( 1 1 )

2. Enhanced Pterin Biosynthesis During Hemopoietic Regeneration Elevated levels of bicpterin and/or of necpterin were observed at the time of hemopoietic regeneration subsequent t o autologous or allogeneic bone marrow transplantation (3,13). In contrast to the process of a l l o g r a f t rejection discussed in the previous paragraph this enhancement of pterin biosynthesis, at least as f a r as necpterin levels were concerned, occured independent from the presence of histocompatibility barr i e r s between the bone marrow donor and recipient (13). In f a c t , simil a r patterns were seen in tumor patients, who subsequent t o l e t a l whole-body irradiation were reconstituted with autologous bone marrow (13). A typical pattern of such a patient i s shown in F i g . 4 .

155

Figure 4: Serum Neopterin and Interferon Levels During Hemopoietic Regeneration Subsequent t o Bone Marrcw Transplant at i on

DAMS

rtfto»· Tfnnsvl nntntlon

We asked the question, whether similar mechanisms involving IFNs or other cytokines such as colon/ stiirulating factors are involved. To t e s t this we have followed both neopterin and I FN levels in several autologous and allogeneic bone marrow transplant recipients. An exaitple i s given in F i g . 4 . Details were also previously reported (13,14). In the majority of patients, detectable levels of circulating IFNs were demonstrated at the time of hemopoietic regeneration and this coincided with the peak necpterin values. This observations supported the view that endogenous IENs might be at least partly involved t o enhanced pterin biosynthesis during hemopoietic regeneration. In contrast no evidence was obtained for the involvement of colony

stiirulating

factors which also peak shortly before hematological reoonstitution.

156

The negative r e s u l t s obtained by in v i t r o stimulation of human nacrophages with GM-CSF are shown in Figure 1. Althcucji involvement c£ endogenous IFNs, which presumably are induced fcy oonfrontation of newly formed lynphocytes with ubiquitous enviromental pathogens i s very l i k e l y , t h i s does not explain the elevation of bicpterin levels observed around t h i s time (3). One thus would rather believe that enhanced p t e r i n biosynthesis during hemopoietic regeneration i s determined by two independent f a c t o r s : an IFN dependent enhancement of early p t e r i n biosynthesis which involves activated Τ - c e l l s and monocyte/ macrophages as well as increased bicpterin content c f iimature hemopoietic c e l l s , c e l l s , vdiich appear in the circulation in large numbers at the time of hemopoietic reconstitution ( 3 ) .

3.Enhanced Pterin Biosynthesis During Endotoxinemia Enhanced pterin biosynthesis during infectious disease i s prinarily a feature of systemic virus disease (for review see 2 ) . Elevated levels were however also observed in a variety of b a c t e r i a l i n f e c t i o n s . By f a r the highest levels were detected in patients suffering from virus d i sease complicated by b a c t e r i a l infection and in septic shock patients ( 4 , 9 ) . A composition of these data i s shown in Figure 5. The l a t t e r observation pronpted the question whether lipcpolysaccharide (LPS) the active principle of endotoxin, which i s known to represent a most potent macrophage stinulator, i s capable t o induce pterin biosynt h e s i s . Previous data fcy C.F.Nathan indicated that LPS also induces neopterin release from human macrophages (12). We subsequently shewed, that LPS induced pterin biosynthesis can not be inhibited t y anti-IFN gaima antibodies (results not shown). This finding indicated a direct action o f LPS on human macrophages. We further investigated, whether IFNs might s e n s i t i z e macrophages t o respond t o endotoxin challenge with increased release of necpterin or of other activation s p e c i f i c cytokines such as tumor necrosis factor (TNF).

157

Figure 5: Necpterin Excreticn in Various Infectious Disease S t a t e s : Upinapu No ot> t ο χ» i η

Ζ c: tu tα. ο — 00

T~E

Μ

•

•

Μ

κ

σ ι ι ο

R—I

ζ -

> en ÜJ

X

ω„ if

CN

- Ρ

ι

ο

C

LU ο :

υ

χ

ο -—ι

Ο ο ο

ο

χ _ι ^

CQ

•

Σ

ZD LU I

Μ CM f—t Ζ ι Ο α: —< UJ ι χ CL Ο Ο

Α

Σ

ο ζ ο ζ — UJ ί Ο

Σ

—

•

BC

Χ ΕΣ >_Ι

c ς:

•

α.

ζ -

α. C.

ο

CN ο

—ι

< Σ CO < _J α.

υ φ

m r-H

> 95% pure as determined by SDS-PAGE. Hydroxylase activity was determined by either a spectrophotometric method (as described in ref. 3) or with a fixed-time assay (14). HPGPC analyses were performed with a column (7.5 χ 600 mm) of TSK 3000 SW (Altex) at ambient temperature (23-25°C) with 0. 1M potassium phosphate, pH 6.8, as the mobile phase. The injection volume was 50 uL for all analyses. The column was calibrated with standard proteins of known molecular weight. Further experimental details can be found in the legends to the various Figures.

Results Two protein peaks were observed when purified rat liver PAH was analyzed by HPGPC

(Fig.l). The major peak corresponded to a molecular

weight of 200000 and the smaller protein peak eluted as Mr-100000. Since PAH consists of identical subunits of Mr-50000 (1), these oligomeric forms of the hydroxylase are presumed to be the tetramer and dimer, respectively. Analysis of 10 different preparations of rat liver PAH, purified by either of two different procedures (6, 13), showed that the tetramer accounts for 78 +/- 6% of the total hydroxylase protein under these conditions. The relative amounts of the tetramer and the dimer were unchanged over a 100fold range of protein concentration, from 0.5 to 50 mg/mL PAH injected for analysis (Fig.l, insert). At concentrations less than 0.5 mg/mL protein, the relative amount of dimer appeared to increase. Limitations of detector sensitivity prevented analysis of samples of less than 0.1 mg/mL hydroxylase

329

time (minutes) FIGURE 1: HPGPC of purified rat liver PAH. 50 uL of a 20 mg/mL solution of PAH was chromatographed, as described in Methods, at 0.5 mL/min, and 0.5 min fractions were collected. Aliquots of these fractions were assayed with 40 uM BH 4 and 2 mM phenylalanine, and with 300 uM 6MPH4 and 10 mM phenylalanine. PAH activity (0) is shown as the ratio % of BH^dependent specific activity to 6MPH4-dependent specific activity (14). Insert: Proportion of tetramer ( • ) and dimer ( | ) as a function of the concentration of PAH injected for analysis.

protein. No evidence for the presence of the monomeric M r =50000 subunit was obtained under denaturing conditions. Preincubation of PAH with dithiothreitol prior to chromatography did not lead to the appearance of monomer. In addition,

only

the monomer was detected by SDS-PAGE in the absence of

thiols. Thus it is unlikely that intersubunit disulfide bonds contribute to the oligomeric organization of PAH. Aliquots of the separated tetramer and dimer were reanalyzed by HPGPC at various times after isolation. The tetramer slowly reverted over a 6h period to a mixture of tetramer and dimer in a proportion identical to that seen with

the original

sample

of PAH. In contrast,

the isolated dimer

remained as a dimer under the same conditions. However, certain conditions can affect the dimer. Preincubation of native PAH with 2 mM phenylalanine and subsequent HPGPC using a mobile phase containing 2 mM phenylalanine

330

phenylalanine in preincubation (mM) FIGURE 2: Effect of preincubation with phenylalanine on the conversion of dimer ( | ) to tetramer (•). Dimeric PAH was incubated for 15 min at 25°C in 0.1M phosphate, pH 6.8, containing the indicated concentrations of phenylalanine. Samples were then analyzed by HPGPC using a mobile phase of 0.1M phosphate, pH 6.8, containing 2 mM phenylalanine. Insert: HPGPC profile of dimeric PAH before ( ) and after ( ) preincubation with 10 mM phenylalanine.

showed

PAH

to be >98%

tetramer

(data not

shown).

Preincubation with

phenylalanine did not affect the elution position of the isolated tetramer. However, similar treatment of the isolated dimer resulted in an increased formation of the tetramer with increasing phenylalanine concentration (Fig. 2). Complete conversion of the isolated dimer to the tetramer was not observed - this may be due in part to the low protein concentration in the isolated dimer pool ("0.3 mg/raL) (cf. insert to Fig. 1). PAH activated by phosphorylation has some of the same characteristics as the enzyme which has been activated by preincubation with phenylalanine (15). Analysis by HPGPC of PAH with a phosphate content of 0.6 mol P^/mol Mr«50000 subunit showed that less than 5% of the hydroxylase protein eluted as the dimer (data not shown). This contrasts with the 25% dimer exhibited by the normal enzyme which contains 0.05-0.2 mol P^/mol subunit (15).

331

8.0 Ο) Ε 7.0 c

ε

6.0

ο μ

5.0

y

"

j0 • / V >

_

g

^

/

:/

-1

-1.2

-1.4

log S

α> C

« 4.0 ο

-

3.0 Ο

Ε 2.0 3L αΟ > 1.0 0

•

"

ÜJ^ i

1 1 0.2 0.4 0.6 0.8 1.0 phenylalanine (mM)

FIGURE 3: BH^-dependent activity of isolated dimeric ( | ) and tetrameric ( • ) PAH as a function of phenylalanine concentration. The oligomeric forms of PAH were isolated as described in the legend to Fig. 1 and immediately assayed with 25 uM BH4 and the indicated concentrations of phenylalanine. Insert: Hill plots of the data from the main Figure, njj for the tetramer was 2.1; njj for the dimer was 1.0.

Kinetics of the oligomeric forms of PAH Both the tetramer and the dimer have identical specific activities when assayed with the synthetic cofactor, 6MPH4. However, the full expression of the regulatory properties and the various activation phenomena to which PAH is subject

is noted only with

the natural cofactor, BH4.

Substantial

differences were observed in the BH^-dependent specific activity of the two oligomeric forms of PAH (Fig. 1); the tetramer has a BH^-dependent specific activity approximately 5 to 6 times that of the dimer. When the resolved oligomeric forms of PAH were assayed immediately after isolation, the BH4dependent activity of the tetramer showed a high degree of cooperativity (njj = 2) in response to variations in phenylalanine concentration (Fig.3), similar to that given by native PAH. The activity of the dimer, on the other hand, was strictly hyperbolic up to about 0.5 mM phenylalanine. The

332

Table 1: Activation of B H ^ d e p e n d e n t dimeric and tetrameric PAH

activity

Dimer

Additions

None 0.2mM phe 0.2mM lysolecithin

13 25 1106

Experiment None 2mM phe 5mM Dhe 20mM phe

56 760 1120 1140

isolated

Tetramer

initial rate (nmol/min/mg)

Experiment 1

of

-fold activation

initial rate (nmol/min/mg)

-fold activation

62

88

1080 1175

17 19

14 20 20

240 2510 2640 2510

11 11 11

2

Aliquots of samples were preincubated for 10 min at 25°C in 0.1 Μ phosphate, pH 6.8, with the indicated addition, then assayed with 50 uM BH^ and 0.2 mM phenylalanine {Experiment 1) or with 38 uM BH 4 and 2 mM phenylalanine {Experiment 2).

BH^-dependent activity of the dimer was however greater than that predicted by the calculated kinetic constants,at higher phenylalanine levels. We feel that

this may be

the result

of the formation of some tetrameric PAH

promoted by these higher levels of phenylalanine (cf. Fig. 2). Preincubation dependent activity

of PAH results

in 10 to 20-fold

increases

in BH4-

(13, 16). The apparent half-maximal concentration of

phenylalanine necessary for this activation at pH 6.8 and 25°C is about 0.1 mM (4, 16). As shown in Table 1, preincubation with 0.2 mM phenylalanine gave a 20-fold increase in the activity of the tetramer, but only a 2-fold increase with the dimer. The extent of activation of the dimer increased with increasing amounts of phenylalanine in the preincubation buffer (Table 1). These increases in activation of the dimer correlated almost exactly with the extent of phenylalanine-promoted tetramer formation under these conditions (Fig. 2). The activation of PAH by the phospholipid lysolecithin differs

from that due to preincubation with phenylalanine

hydroxylase

is

desensitized

in

the

presence

of

in that the

lysolecithin

(2).

In

addition, the phospholipid induces a gross conformational change in enzyme structure (17) that is not observed upon preincubation of the enzyme with phenylalanine (12). Table 1 shows that both dimeric and tetrameric PAH are

333

activated by lysolecithin. Note that the BH^-dependent specific activity of both species is essentially identical in the presence of lysolecithin. We have been unable to determine the oligomeric nature of PAH in the presence of lysolecithin levels

of

due to interference

lysolecithin

used.

from phospholipid micelles at the

However,

PAH which

has

been

chemically

modified by reaction with NEM is in an activated and desensitized form, kinetically identical to the phospholipid treated enzyme (3). NEM-modified PAH exists entirely as dimers under our conditions of HPGPC (data not shown).

Discussion

The BH4-dependent activity of rat liver PAH shows positive cooperativity with respect to variations in phenylalanine concentration (2). Such kinetic data have been used to implicate the presence on PAH of distinct catalytic and regulatory (or activator) sites for phenylalanine (16, 18). The first direct evidence in support of this proposal was the finding that native PAH binds more than 1 mol of phenylalanine per Mr=50000 subunit (3). Interpretation

of

the

non-integral

value

of

1.5

phe/subunit

was not

possible at that time, given the paucity of information concerning the subunit composition of PAH. However, others had proposed that PAH possessed 1 regulatory site /subunit (9). A number of subsequent findings in addition to the present work have allowed us to reassess the subunit organization of the catalytic and regulatory sites for phenylalanine on PAH: (i) The Mr=50000 monomeric subunits of PAH have been shown to be identical in primary structure (1). (ii) With BH^ as cof actor, the presence of tryptophan results in the desensitization of the response of PAH to phenylalanine concentration, but does not affect V m a x . The binding of phenylalanine to PAH in the presence of tryptophan is reduced to 1 phe/subunit from 1.5 phe/subunit (4). (iii) Studies of the quenching of the intrinsic fluorescence of PAH by BH4 demonstrate a binding ratio of 1 mol of BH^/mol of Mr—50000 subunit (12). Since the tetrahydropterin is essential for hydroxylation (19), we assume that each subunit must possess a single catalytic site. This assumption is consistent with (ii), and with the observation that PAH, desensitized yet

334 activated by modification with NEM, binds 1 phe/subunit (3). In addition, these observations indicate that conditions which lead to desensitization of PAH (i.e., the abolition of positive cooperativity with respect to phenylalanine) also result in the apparent disappearance of 0.5 sites for the binding of phenylalanine per subunit. It seems likely therefore that each monomeric

Mr—50000

subunit of PAH has 1 catalytic

site and the

equivalent of or potential for 0.5 regulatory (or activator) site. Since no evidence for the presence of monomeric Mr-50000 subunit was obtained in the present study, we consider the minimum reactive unit of PAH to be the dimer. The data which describe the the binding of phenylalanine to PAH (3, 4) are consistent with the idea that this dimer might possess 3 binding sites for phenylalanine - 2 catalytic sites, and 1 regulatory site. However, we do not think that this is an accurate picture of the organization of these sites. The regulatory site is presumably involved in the expression of the complex BH^-dependent kinetics of PAH with respect to phenylalanine, and

is necessary for the activation of PAH noted upon

preincubation of the enzyme with phenylalanine. The dimer, however, shows a strictly non-cooperative response to variations in phenylalanine concentration; only the tetramer shows kinetic cooperativity (Fig. 3). The dimer can apparently be activated by preincubation with phenylalanine, but the extent of activation is strongly correlated with the amount of tetramer formed under the conditions of preincubation (Fig. 2; Table 1). Thus at least part, and perhaps all, of the activation of the dimer induced by preincubation with phenylalanine is due to the phenylalanine-promoted conversion of dimers to tetramers. A simple model for the possible organization of the regulatory binding sites for phenylalanine on PAH is illustrated in Figure 4. Phenylalanine hydroxylase

is

an

oligomeric

protein

composed

of

subunits

which are

identical in primary structure. Each subunit possesses 1 catalytic site and the equivalent of, or the potential for, 0.5 regulatory site. The minimum active catalytic unit of PAH is a dimer. These dimers in the native enzyme exist in equilibrium with tetramers which are thus, in effect, "dimers of dimers". When two dimers associate in the proper manner to form a tetramer, 2 additional binding sites for phenylalanine are "created", in addition to the 4 catalytic sites already in existence. These two newly-formed sites are the regulatory or activator sites for PAH, and may exist at the

335

active

low activity phosphorylation (?)

QQ

Phe

N-ethylmaleimide

active FIGURE 4: Scheme for the oligomeric forms and organization of the regulatory binding sites for phenylalanine on PAH. Details are outlined in the text.

interface of the dimer-dimer interaction. In this model, both the native dimer

and

the

native

tetramer

(circular

figures) have

low

intrinsic

catalytic activity. The binding of phenylalanine to the regulatory sites (this binding is denoted by the shaded ovals), which exist in complete form only in the tetramer, results in (i) stabilization of the tetramer, and (ii) some conformational response in PAH which results in activation of the enzyme. Conformational changes in PAH due to the binding of phenylalanine are well documented

(12, 13). Since the dimer does not possess intact

regulatory sites for phenylalanine, the activity of this species remains low, with no observed cooperativity. The NEM-modified PAH is shown as a dimer in a different conformation than the native dimer, since it is an activated form of PAH. The dimeric nature of NEM-PAH is consistent with both

the absence

of kinetic

cooperativity

and

the

decrease

in

total

phenylalanine binding exhibited by this activated enzyme. Support for the concept of 2 regulatory binding sites for phenylalanine/tetramer has been obtained from recent studies concerning the interaction of a monoclonal antibody with PAH. This antibody binds to PAH only after the enzyme has been incubated with phenylalanine (20). Therefore, this antibody interacts with the tetrameric form of PAH. Binding of the

336 antibody inhibits PAH activity

in a noncompetitive manner

(21) and is

therefore presumably not binding to the catalytic region of the hydroxylase. The interaction results in the abolition of the cooperative response of BH^-dependent PAH activity to variations in phenylalanine concentration (M.A. Parniak, unpublished data), thus the antibody may be interacting with PAH at or near the regulatory region of the enzyme. This antibody binds to 2 sites over the 4 identical subunits of tetrameric PAH. The model presented in this report allows for 1 catalytic site/monomer of PAH, and the data indicate that the regulatory or activator site for phenylalanine is not necessary for the expression of low levels of catalytic activity in the dimer. Thus it is possible that the monomer could be catalytically active, if it could be formed and stabilized under nondenaturing conditions.

Acknowledgements M.A.P. is a Qu£bec. This Dr. Seymour enthusiastic

Chercheur-Boursier of the Fonds de la Recherche en Santfe du work was initiated while the author was in the laboratory of Kaufman, NIMH, Bethesda, MD. I gratefully acknowledge his support, instruction, and stimulating insight and discussions.

References 1. Iwaki, M., Parniak, M.A., Kaufman, S. (1985) Biochem. Biophys. Res. Commun. 126:922-932 2. Fisher, D.B., Kaufman, S. (1973) J. Biol. Chem. 248:4345-4353 3. Parniak, M.A., Kaufman, S. (1981) J. Biol. Chem. 256:6876-6882 4. Phillips, 259:271-277

R.S.,

Parniak, M.A., Kaufman,

S.

(1984) J.

Biol. Chem.

5. Fersht, A. (1977) Enzyme Structure and Mechanism. W.H. Freeman, San Francisco 6. Kaufman, S., Fisher, D.B. (1970) J. Biol. Chem. 245:4745-4750 7. Doskeland, A.L., Jones, Τ., et al (1982) Neurochem. Res. 7:407-421 8. Nakata, Η., Fujisawa, Η. (1980) Biochim. Biophys. Acta 614:313-327 9. Shiman, R. (1980) J. Biol. Chem. 255:10029-10032

337

10. Tourian, Α. (1971) Biochim. Biophys. Acta 242:345-354 11. Ayling, J.E., Pirson, W.D., et al (1974) Biochemistry 13:78-85 12. Phillips, R.S., Parniak, M.A., Kaufman, S. (1984) Biochemistry 23:38363842 13. Shiman, R., Gray, D.W., Pater, A. (1979) J. Biol. Chem. 254:11300-11306 14. Hasegawa, Η., Kaufman, S. (1982) J. Biol. Chem. 257:3084-3089 15. Parniak, M.A., Hasegawa, Η. , et al Commun. 99:707-714

(1981) Biochem. Biophys. Res.

16. Shiman, R., Gray, D.W. (1980) J. Biol. Chem. 255:4793-4800 17. Abita, J.P., Parniak, M.A., Kaufman, S. (1984) J. Biol. Chem. 259:1456014566 18. Dhondt, J.L. , Dautrevaux, Μ., et al (1978) Biochimie 60:787-794 19. Kaufman, S. (1959) J. Biol. Chem. 234:2677-2682 20. Jennings, I.G., Russell, R.G.McR., et al (1986) Biochem. J. 235:133-138 21. Parniak, M.A., Jennings, I.G., Cotton, R.G.H. (1986) in Chemistry and Biology of Pteridines (Cooper, B.A., Whitehead, V.M., eds.), Walter de Gruyter, Berlin-New York, pp. 359-362

IMMUNOCHEMICAL STUDIES OF PHENYLALANINE HYDROXYLASE

R. G. H. Cotton, I. G. Jennings, W. McAdam and S. Smith

Olive Miller Protein Laboratory, Murdoch Melbourne, Australia

Institute,

Introduction

Phenylalanine hydroxylase

(PH) is a complex enzyme which has

three substrates phenylalanine, tetrahydrobiopterin and oxygen and its enzyme activity is activated by phenylalanine, phosphorylation, N-ethyl maleimide, and phospholipids especially lysolecithin

(1).

We have initiated a study to

assess the ability of monoclonal antibodies, in combination with traditional protein chemistry, to define the

structural

regions of PH polypeptide responsible for the catalytic functions of the enzyme.

A monoclonal antibody

(MAb) which

bound the catalytic site of the enzyme might be expected to show the following characteristics

: (a) binding of the MAb

to PH at equimolar concentration would completely enzyme activity

inhibit

(b) binding of MAb to PH would be inhibited

by substrate or enzyme inhibitors

(c) MAb would bind

tyrosine

and tryptophan hydroxylases which share a common substrate with PH and also with other enzymes which have some common substrates

(d) M A b 1 s epitope would be a highly conserved

sequence in PH and thus MAb would recognise PH from a wide range of species in the phylogenic

spectrum.

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

340 Results and Discussion

A series of eleven monoclonal antibodies

(2) recognised at

least five different antigenic regions on PH and were tested for their ability to satisfy the above criteria.

The results

are summarized in Table 1.

Table 1 Group

A

Β C

D Ε

+

Antibody

PHI PH5 PH8 PH7 PH2 PH3 PH10 PHI 2 PH9 PH11 PH6

Effect on Activity 0 -59 +43 -19 +29 + 100 + 100 +33 +33 + 12 +16 -6

Species Reactivity* Η Η Η Η Η Η Η Η Η Η Η

Μ Μ Μ Μ Μ Μ Μ Μ Μ Μ Μ

Ligand

R Phe R Lyso R & others Lyso R P04 Lyso R Lyso R Lyso R Lyso none none R Lyso

χ

(+) (-) (-) (+) (-) (-) (-) (-)

(-)

Antibodies are grouped according to their ability to compete with each other for binding to PH ° Percentage effect on enzyme activity were calculated by using the enzyme activity with no antibody present as 0%; (+) stimulation (-) inhibition of enzyme activity. * Η, M, R designate human, monkey and rat liver PH respectively. χ effect on MAb binding of phenylalanine (PHE), lysolecithin (Lyso) and PO^ (covalently bound phosphate) when bound to PH (+)/ (-) designate promotion and inhibition of MAb binding respectively. It can be seen from Table 1 that no MAb has satisfied the criteria detailed earlier for active site recognition.

PHI MAb inhibited the enzyme by 59%, required for its binding

phenylalanine

(%-maximal phenylalanine concentration is

similar to that required for activation) it has been shown

(2).

In addition,

(3, in preparation) that the MAb inhibition

is non-competitive and thus PHI may be recognising a response

341

of PH to a c t i v a t i o n by

phenylalanine.

PH7 M A b only b i n d s to p h o s p h o r y l a t e d PH is l o c a t e d w i t h i n the

(4) a n d its

epitope

sequence

15

19 Leu-Ser-Asp-Phe-Gly. PO 4

PH7 M A b h a s little e f f e c t o n e n z y m e a c t i v i t y w h i c h is c o n s i s t e n t w i t h o t h e r f i n d i n g s t h a t the p h o s p h o r y l a t i o n is n o t c o n t a i n e d w i t h the c a t a l y t i c

site

site.

The b i n d i n g of a n u m b e r o f M A b s ' to PH is a f f e c t e d b y lysolecithin.

H o w e v e r only P H 2 , 3 a n d 5 show a

stimulation

o f similar m a g n i t u d e to l y s o l e c i t h i n w i t h t h e s e M A b s

also

m i m i c k i n g the c h a n g e s by l y s o l e c i t h i n to the PH e n z y m e kinetics

(2).

The e p i t o p e o f t h e s e M A b s has n o t b e e n

l o c a l i z e d b u t is likely to be in the Ν t e r m i n a l bromide peptide

finally

cyanogen

(unpublished).

PH8 M A b r e a c t s w i t h PH in W e s t e r n b l o t s of l i v e r from a w i d e range of s p e c i e s

extracts

(man, r a t , d o g , p i g , c o w ,

c h i c k e n , fish, frog b u t n o t s q u i d or b a c t e r i a )

(5).

koala,

PH8

has

b e e n s h o w n to b i n d t y r o s i n e h y d r o x y l a s e by E L I Z A a n d t r y p t o p h a n h y d r o x y l a s e by s o l u t i o n r e a c t i o n immuno h i s t o c h e m i c a l

(6).

In a d d i t i o n

s t a i n i n g of b r a i n t i s s u e h a s s h o w n PH8

reacts with tyrosine and tryptophan hydroxylase has b e e n u s e d to study the d i s t r i b u t i o n of n e u r o n e s in the h u m a n b r a i n stem

(6) a n d

it

serotinergic

(in p r e p a r a t i o n ) .

PH8

MAb

e p i t o p e h a s b e e n i d e n t i f i e d as 138 Gly-Ala-Glu-Leu-Asp-Ala-Asp-His-Pro-Gly-Phe-Lys-Asp-Pro 154 -Val-Tyr-Arg

; (5) a r e g i o n s u b s t a n t i a l l y c o n s e r v e d

tyrosine hydroxylase

(7) .

in

342

PH9 has b e e n s h o w n to r e c o g n i s e the Ν t e r m i n a l p e p t i d e o f h u m a n PH

tryptic

(in p r e p a r a t i o n ) .

T h e f i g u r e shows the d i s t r i b u t i o n of the k n o w n b i n d i n g

sites

of the M A b s PH 2, 3, 7, 8 a n d 9. CNBR CLEAVAGE t

PO. Li "9

7

"ΊΓ




2,3 R e c e n t l y w e h a v e i s o l a t e d a s e r i e s of a n t i b o d i e s w h i c h m o r e c l o s e l y fit the c r i t e r i a for a c t i v e site r e a c t i v e One a n t i b o d y b i n d s to PH, h u m a n d i h y d r o p t e r d i n e

antibodies.

reductase,

a n d E. coli d i h y d r o f o l a t e r e d u c t a s e a n d its b i n d i n g to PH is i n h i b i t e d by the p r e s e n c e o f 6,7 d i m e t h y l 7,8

dihydropterin.

T h e i d e n t i f i c a t i o n o f this M A b ' s e p i t o p e o n PH is u n d e r w a y .

References 1.

S h i m a n , R. In P t e r i n s a n d F o l a t e s , V o l . 1 Eds B l a k l e y , R . L . , B e n k o v i c , S.J. J. W i l e y and S o n s , 1984.

2.

J e n n i n g s , I. G . , R . G . M c R . R u s s e l l , W . L . F . A r m e r g o a n d R. G. H. C o t t o n . B i o c h e m . J . (1986) 235 133-138.

3.

P a r n i a k , Μ . Α . , I. G. J e n n i n g s a n d R. G. H. C o t t o n (1986) p . 3 5 9 - 3 6 2 in C h e m i s t r y a n d B i o l o g y of P t e r i d i n e s , 1986. E d s B. A . C o o p e r a n d V . M. W h i t e h e a d . Published W. de G r u y t e r .

4.

S m i t h , S. C . , W. J. M c A d a m , Β. E. K e m p , F. J. M o r g a n a n d R. G. H. C o t t o n (1986) B i o c h e m . J. (in p r e s s ) .

5.

H a a n , Ε. Α . , I. G. J e n n i n g s , A. C. C u e l l o , H. N a k a t a , C. W . C h o w , R. K u s h i n s k y , J. B r i t t i n g h a m a n d R. G. H. C o t t o n (1986) B r a i n R e s e a r c h (submitted).

6.

M a n u s c r i p t in p r e p a r a t i o n .

7.

Grima, B., Lamouroux A, Blanot F., Biguet N.F. M a l l e t J. (1985) P . N . A . S . 82 617-621.

and

INFLUENCE OF SUBSTRATES AND COFACTORS ON TYROSINE HYDROXYLASE

D.M. Kuhn Department of Psychiatry, Lafayette Clinic, Wayne State University School of Medicine, Detroit, Michigan 48207 USA

Introduction

Tyrosine hydroxylase (TH) is the initial and rate limiting enzyme in the biosynthetic pathway for the catecholamines dopamine, norepinephrine, and epinephrine. It was recognized some time ago that the activity of TH in adrenal and brain extracts was dramatically increased upon the addition of reduced pterins (1,2). Since these initial studies, a large amount of information on the pterin dependent enzymes TH, phenylalanine hydroxylase, and tryptophan hydroxylase has emerged concerning the mechanisms by which their level of activity is controlled. Most of these experiments were designed to study posttranslational influences such as phosphorylation, or the effects of various drugs on enzyme activity and little attention has been directed at the role that the pterin cofactor might play, other than the most obvious one of an essential cofactor in altering the activity of the hydroxylase enzymes. With the discovery of variant forms of hyperphenylalanemia (3-5) which involved primary defects in the enzymes of tetrahydrobiopterin (BHu) synthesis and not in phenylalanine hydroxylase, a vastly different etiology for practically all disorders involving the biogenic amine neurotransmitters and the aromatic amino acid hydroxylases was suggested. These disorders range from inborn errors in metabolism such as hyperphenylalanemia, mentioned above, to many psychiatric and neurologic disorders (see Levine (6) this volume). The studies of Kettler et al (7) were perhaps the first to demonstrate that the parenteral administration of BH4 to rats caused an increase in the cerebral levels of the cofactor and an increase in catecholamine

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

344 turnover. These results formed an important basis for the therapeutic treatment with BH« of certain diseases involving deficits in neurotransmitters since they offered the possibility that BHa could produce increases in neurotransmitter levels. The administration of BH4, like any therapeutic agent, to animals or man presupposes that the compound will reach its site of action and result in an increase in the activity of the hydroxylases for which it is the essential cofactor. In general, the most accessible hydroxylase has been phenylalanine hydroxylase because of its location in the liver. The localization of tryptophan hydroxylase and TH principally in brain restricts access of BE» to these enzymes to some extent but structure activity studies with the cofactor have revealed that some congeners can penetrate the blood-brain barrier (8). Once the cofactor reaches the hydroxylase enzyme it can interact with it to result in increased activity and increased production of the neurotransmitters dopamine, norepinephrine, and serotonin. The exact mechanism by which BH4 interacts with the hydroxylase enzymes is gradually becoming more completely understood as the reaction mechanisms of the enzymes are elucidated. Studies with purified phenylalanine hydroxylase have allowed impressive advances in our understanding of how BH4 interacts with this enzyme (9,10). Other equally interesting experiments have discovered numerous means by which BH« can interact with phenylalanine hydroxylase to influence its structure and level of activity by mechanisms which are not strictly limited to a cofactor role (11,12). In an attempt to determine if Bftj can interact with TH as it does with phenylalanine hydroxylase we undertook a series of experiments on BH4-TH interactions which began with purification of the hydroxylase enzyme to homogeneity. TH is a very unstable enzyme but sufficient quantities were purified from cultured PC12 cells to allow studies on its catalytic properties (13). Our initial experiments indicated that if the enzyme was preincubated with BH4, catalytic activity was inhibited in subsequent assays (14). The other cosubstrates for TH, tyrosine and oxygen, did not inactivate the enzyme under the same incubation conditions. The ability of the cofactor to inactivate TH was both time and temperature dependent and was restricted to reduced pterin species.

345 Solutions of BH4 are composed of two diastereoisomers referred to as the 6R and 6S forms (15). The isomers were separated and tested individually for their ability to inactivate TH and we observed that the natural 6R form was far more potent than the 6S isomer. Finally, we tested the pterin analog 6-methyl-5-deazatetrahydropterin and the pyrimidine 2,5,6triamino-4-pyrimidinone for their ability to mimic the BfU induced inactivation of the enzyme and observed these compounds were almost equipotent with BH4. These results were somewhat surprising since these two

compounds have little if any cofactor activity but they are capable

of inhibiting in vitro hydroxylase activity by virtue of their ability to compete for BIU (16). In order to determine further if BH4 was directly inactivating TH by altering the enzyme we carried out additional studies on reversal of the inactivation. Furthermore, we tested the ability of the substrates and cofactor of TH to influence the phosphorylation of TH by the cyclic 3',5' adenosine monophosphate (cAMP) -dependent protein kinase. These studies were important because phosphorylation of TH activates the enzyme (17,18) and this activation is mediated by a reduction in the km of the enzyme for BH4. Since BH4 can dramatically influence the phosphorylation of phenylalanine hydroxylase (12,18) by protein kinases we felt it was important to determine if BH4 could similarly alter the phosphorylation of TH. Such an effect could have an influence on any therapeutic properties of BH4 if the cofactor could alter an important control process for TH.

Results Purified TH was first incubated with 0.5 mM BH« for 30 min and the mixture was either dialyzed against 500 volumes of 50 mM potassium phosphate buffer pH 7.2 containing 40X glycerol and 0.1 mM EDTA or Chromatographed over a small column of Dowex 50 cation exchange resin. The results are shown in Table 1.

346 Table 1

Effects of dialysis and Dowex 50 chromatography on the inactivation of TH by BB» % Control

Dialysis

Dowex 50

Control

92

143

115

BH4

51

147

53

Preincubation with Bfti resulted in a 50* reduction of ΊΉ activity as previously observed (14). Upon dialysis, the control preparation was increased in activity as was the BH4 inactivated enzyme. On the other hand, cation exchange chromatography did not reverse the inactivation of TH. These results suggest that the inactivation produced by Bftj is reversible and that incubation of the enzyme in the presence of BH4 (and without tyrosine) generates an inhibitory substance. The incubations were repeated in experiments where tyrosine was added in increasing concentrations and the effects are included in Table 2. Table 2 Effects of tyrosine on the BEi-induced inactivation of tyrosine hydroxylase

Tyrosine (mM)

Tyrosine Hydroxylase (X Control)

0 0.025 0.05 0.075 0.10 0.20

48 50 74 87 100 120

It can be seen that when the incubations with BH4 are carried out in the presence of tyrosine, the inactivation by the cofactor was prevented in a concentration dependent manner. These conditions progressively approximate the normal in vitro catalytic conditions and demonstrate that the inhibition by BH4 can occur only when the enzyme is exposed to

347 the cofactor under non-catalytic conditions.

Thus, it appears that when

the normal oxidation-reduction of BH« takes place as it does during catalysis, the enzyme is obviously not inactivated. However, in the absence of tyrosine, exposure of TH to BH» results in the formation of an inhibitory product and this product does not seem to be an oxygen or superoxide radical (14). Phosphorylation of TH activates the enzyme an causes a reduction in the km of the enzyme for BH4. It has also been shown that incubation of phenylalanine hydroxylase with BH4 can dramatically alter the effects of phosphorylation on this enzyme and these experiments were repeated with purified TH. The enzyme was first incubated with BH4 (with or without tyrosine) to inactivate catalysis and the catalytic subunit of the cAMPdependent protein kinase was added in the presence of (y 32 P)ATP. It can be seen in Figure 1 that the incorporation of

32

P into TH was not

altered by BH4. Other lanes indicate that tyrosine or BH» plus tyrosine are also without effects on the phosphorylation of TH.

Fig. 1

Effects of BH4, tyrosine, or BH4 plus tyrosine incubation on the phosphorylation of TH. Purified preparations of the enzyme were preincubated (from left to right) with 1) buffer, 2) 0.5 nM BH«, 3) 1.0 nM ΒΗ», 4) 0.1 mM tyrosine, 5) 0.5 nM BR» + tyrosine, or 6) 1.0 nM BH« + tyrosine and after 30 min incubation the catalytic subunit of cAMP-dependent protein kinase was added.

348 After a 5 min reaction, samples were prepared for SDSpolyacrylamide electrophoresis. Gels were stained and dried followed by exposure to x-ray film to determine the extent of incorporation of

32

ρ into TH.

It appears from the results in Fig. 1 that the catalytic status of TH does not influence its ability to serve as a substrate for the exogenous protein kinase. Enzyme samples with less than 50* of their catalytic capacity (lane 2) or with complete activity were phosphorylated to the same extent.

Discussion Previous experiments with BH4-TH interactions under non-catalytic conditions were directed at learning more about the processes by which the cofactor could influence the enzyme apart from serving as an essential cofactor (14). It is clear from the present data that Blk can inactivate TH in the absence of tyrosine but this does not represent a normal in vivo condition. However, if large doses of BH4 resulted in the elevation of brain cofactor levels it is conceivable that TH activity could be reduced, especially if the levels of tyrosine were also less than normal. Recent experiments on the mechanism of action of purified TH (19) indicated that BH4 can influence the enzyme in a fashion similar to that reported above. Incubation of TH with stoichiometric amounts of BH4 (1.0 uM) for various times revealed that enzyme activity was reduced by 50* within 1 min of exposure of the enzyme to BH4, and was further reduced to approximately 20* of control after 30 min. Dix et al (19) suggested that a transient hydroxylating species was being generated by TH from BH4 and oxygen and these results confirmed their hypothesis. Furthermore, the loss of activity under the incubation conditions described could not be prevented with catalase, suggesting that hydrogen peroxide was not the inactivating species (19). These data concerning TH-BH4 interactions at very low concentrations of BH4 are in agreement with our previous research (14) and suggest several explanations for the BH4-induced inactivation of TH. First, the BH4

349 could oxidize the enzyme-bound iron (19) which would lead to reductions in activity. It has been demonstrated that the fluorescence properties of TH are altered by BH4 (19). Second, exposure of the enzyme to BH4 in the absence of tyrosine might lead to the production of a chemical species which interferes with subsequent catalytic activity. The reversal of the inactivation of TH by dialysis suggests the existence of such a species and makes less likely the possibility that the enzyme is being directly altered. Additional research will be necessary to identify the inactivating compound. The data with phosphorylation of TH indicate that the cofactor does not influence the ability of the enzyme to serve as a substrate for cAMPdependent protein kinase. TH was phosphorylated to the same extent regardless of its catalytic status. Thus, the enzyme can be phosphorylated in the presence of BH4, even after inactivation of TH with the cofactor. The activation of TH by phosphorylation represents an important control mechanism for the enzyme and it seems clear that the substrates and cofactor do not modulate the actual phosphorylation of TH as they do with phenylalanine hydroxylase, where BH4 can significantly diminish the phosphorylation of phenylalanine hydroxylase (12,18). While BH4 does not alter the phosphorylation of TH, it has been reported that the phosphorylated form of TH is less stable than the non-phosphorylated form (19,20) and BH4 can further enhance the destabilizing effects of phosphorylation (21). In summary, BH4 does not directly alter the phosphorylation of TH but it can lead to the enhanced inhibition of the activated form of the enzyme. It is not possible to determine the status of activation of TH in vivo but it can be hypothesized that it is functioning at less than optimal levels of activity in neuro-psychiatric disorders where transmitter deficits are thought to contribute to the cause of the disease. In this fashion, elevation of cerebral levels of BH4 would not be expected to reduce TH activity but hopefully produce an increase.

350 References 1.

Brennenman, A.R. and S. Kaufman. 1964. Biochem. Biophys. Res. Comm. 17, 177.

2.

Nagatsu, Τ., M. Levitt, S. Udenfriend. 1964. Biochem. Biophys. Res. Comm. 14, 543.

3.

Kaufman, S., N.A. Holtzman, S. Milstein, I.J. Butler, A. Krumholtz. 1975. N. Eng. J. Med. 293, 785.

4.

Niederwieser, Α., Ch. Curtius, 0. Bettoni, J. Bieri, B. Schircks, M. Viscontini, J. Schwab. 1979. Lancet 1, 131.

5.

Niederwieser, Α., Ν. Blau, Μ. Wang, Ρ. Joller, Ν. Atares, J. Cardesa-Garcia. 1984. Eur. J. Pediatr. 141, 208.

6.

Levine, R.A. 1987. In: Unconjugated Pterins and Related Biogenic Amines (H.-C. Curtius, N. Blau, and A. Niederwieser, eds.). Walter de Gruyter & Co.

7.

Kettler, R., G. Bartholini, and A. Pletscher. 1974. Nature 249, 476.

8.

Levine, R.A., H.-C. Curtius, B. Schircks: J. Pharmacol. Exp. Ther. (in press)

9.

Benkovic, S.J., L.J. Slieker, S.C. Daubner, L.F. Couortney, T.A. Dix, S.O. Pember, L.M. Bloom, C.A. Fierke, R.J. Mayer, J.-T. Chen, K. Taira. 1986. In: Chemistry and Biology of Pteridines 1986 (B.A. Cooper and V.M. Whitehead, eds.). Walter de Gruyter, Berlin-New York, p. 13.

10.

Shiman, R. 1985. In: Folates and Pterins, Vol. 2 (R.L. Blakley and S.J. Benkovic, eds.). John Wiley & Sons, p. 179.

11.

Phillips, R.S., I.Iwaki, S. Kaufman. 1983. Biochem. Biophys. Res. Comm. 110, 919.

12.

Phillips, R.S. and S. Kaufman. 1984. J. Biol. Chem. 259, 2474.

13.

Kuhn, D.M. and M.L. Billingsley: Neurochem. Int. (in press)

14.

Kuhn, D.M. and W. Lovenberg. 1983. Biochem. Biophys. Res. Comm. 117. 894.

15.

Bailey, S.W. and J.E. Ayling. 1978. J. Biol. Chem. 253, 1598.

16.

Moad, G., C.L. Luthy, P.A. Benkovic, S.J. Benkovic. 1979. J. Amer. Chem. Soc. 101, 6068.

351 17.

Kuhn, D.M. and W. Lovenberg. 1983. In: Handbook of Neurochemistry, Vol. 4 (A. Ljatha, ed.). Plenum Press, New York, p. 133.

18.

Kaufman, S. and E.E. Kaufman. 1985. In: Folates and Pterins, Vol. 2 (R.L. Blakley and S.J. Benkovic, eds.). John Wiley & Sons, p. 251.

19.

Lazar, M.A., R.J.W. Truscott, J.D. Raese, J.D. Barchas. 1981. J. Neurochem. 36, 677.

20.

Vrana, K.E. and R.Roskoski. 1983. J. Neurochem. 40, 1692.

21.

Roskoski, R., T.P. Finney, D.J. Täte, K.E. Vrana. 1983. Fed. Proc. 42, 2022.

MECHANISM OF ACTIVATION OF TRYPTOPHAN 5-MONOOXYGENASE BY MERCAPTIDES

H. Hasegawa

and

A. Ichiyama

Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan

Introduction Tryptophan 5-monooxygenase has been assayed in the presence of mercaptides, ferrous iron, and catalase in addition to the substrates, tryptophan and tetrahydropterin under aerobic conditions.

Although the employment of mercaptide,

iron and

catalase is important for obtaining reproducible and high activity, they have been more-or-less empirically used.

Since

the first demonstration of an activation of bovine pineal tryptophan 5-monooxygenase by an anaerobic incubation with dithiothrei tol (DTT) (1), we have been working on the mechanism of mercaptide activation of the enzyme though the source of enzyme was switched from bovine pineal gland to mouse mastocytoma.

The present work is on the same line.

Prelimi-

nary accounts on the effects of catalase and iron were presented before (2,3).

It was possible that the high concen-

tration of DTT in our activation procedure changes conformation of the enzyme by opening disulfide linkage(s).

Then this

possibility was examined by determining the number of sulfhydryl groups

(SH) of the e n z y m e

before

and

after

the

activation.

Materials and Methods N-( 7-Dimethylamino- 4-methy 1 coumarinyl )-maleimide (DACM) was

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

354

used as a fluorogenic reagent for the determination of free SH in the enzyme protein.

This reagent was purchased from Wako

Chemical Industries, Tokyo, Japan, and was dissolved in acetonitryl and stored at -80° until use. Tryptophan 5-monooxygenase was purified essentially according to Nakata and Fujisawa (4) from a mouse mastocytoma, P-815. The purity of the enzyme used was more than 98% as judged by SDS-gel electrophoresis. Reduction of tryptophan 5-monooxygenase with DTT was performed in the same way as that for the activation of the enzyme under N2-

The standard mixture contained 15 m M DTT in 50 mM Tris-

acetate (pH 8.1) as the minimum components. conditions were achieved

The anaerobic

in a Thunberg tube connected to

vacuum line and nitrogen container by replacing air in it with N2.

This procedure was essentially the same as the original

method developed for the activation of bovine pineal tryptophan 5-monooxygenase (1), though catalase and iron salt were omitted because they have been known not to be essential as long as the activity assay was performed just after opening the Thunberg tube and the compounds were supplied into the reaction mixture (3). For determining SH of native enzyme, the enzyme was kept in a stabilizing buffer, 50 mM Tris-acetate (pH 7.5) containing 0.1 Μ KCl,

10% glycerol,

0.05% Tween 20.

Reduced enzyme was

subjected to the SH assay directly as a mixture with DTT used for the reduction as described above.

Denaturation of the

enzyme was performed with 6 Μ guanidine-HCl in the stabilizing buffer to give non-reduced denatured protein and in the 15 mM DTT solution under N2 to give fully reduced denatured protein. The proteins (30-50 pg in 200 p i ) were applied on a column of Sephadex G-2 5 (bed vol. 2.2 ml) equilibrated with 50 pM DACM in 50 m M Na-phosphate (pH 6.8) containing 10% glycerol or 6M guanidine-HCl for native or denatured proteins,

respectively.

Fractions, 200 pi each, were collected by stepwise elution. The peak fractions of protein were acidified by addition of 1/5 vol of 100%-formic acid.

A 200 pi aliquot of the solution

355 was a p p l i e d t o a n o t h e r Sephadex G-25 column o f t h e same d i m e n sion e q u i l i b r a t e d

of

the

e l u a t e f r o m t h e s e c o n d c o l u m n (200 ^il e a c h ) w e r e d i v i d e d

for

assays of

with

10% f o r m i c a c i d .

p r o t e i n and f l u o r e s c e n c e .

Both samples w e r e

l i z e d and s u b j e c t e d t o e a c h a s s a y . fluorescence,

the l y o p h i l i z e d

Li-dodecy1sulfate

in

The f r a c t i o n s

lyophi-

For d e t e r m i n a t i o n of

proteins

were d i s s o l v e d

0.1 Μ L i - b o r a t e

(pH 8 . 5 )

i n 6 mM

and s t o o d

room t e m p e r a t u r e f o r a t l e a s t o n e d a y i n t h e d a r k t o the spontaneous r i n g - o p e n r e a c t i o n ured

at

390

nm

for

excitation

(5).

at

ensure

F l o r e s c e n c e was meas-

and

476

nm

for

emission.

P r o t e i n was d e t e r m i n e d by t h e m e t h o d o f L o w r y e t a l BSA as t h e

the

(6)

with

standard.

Results The

content

of

free

SH o f

m e a s u r e d b e f o r e and a f t e r activation alkylating

of

tryptophan the

5-monooxygenase

anaerobic

incubation

t h e e n z y m e w i t h 15 mM DTT ( 3 ) .

reagent,

DACM,

was employed

mixing

the

reagent

with

pico-mole

as t h e

high

amount o f

for

labeling

total

cysteine

content,

was a l s o s u b j e c t e d it

possible

separation

the

of

with

To

avoid

of

the

mix-

In order

d e n a t u r e d enzyme i n

to the determination.

quick

labeling

of

DTT,

Sephadex

to obtain

the enzyme from t h e bulk o f

the

6 Μ guanidine-HCl

This procedure

the

and

SH ( 5 ) .

concentrations

50 jiM DACM.

probe

conditions

t u r e o f t h e e n z y m e and DTT was a p p l i e d t o a c o l u m n o f G-25 e q u i l i b r a t e d

the

A fluorogenic

w h i c h r e a c t s w i t h f r e e SH q u i c k l y a t n e u t r a l e n a b l e s us t o d e t e r m i n e

was

free-SH DTT.

just

made after

Subsequently,

the t r a c e o f DTT-DACM a d u c t s which c o n t a m i n a t e d

in the

f r a c t i o n and f r e e DACM w e r e removed by a n o t h e r

gel-filtration

t h r o u g h a S e p h a d e x G-25 c o l u m n e q u i l i b r a t e d acid.

Figure

1 shows

chromatography. the

same b e t w e e n

enzyme

reduced

the

elution

The amount o f the

fully

under

the

w i t h 10% f o r m i c

profiles

labeled

activating

of

SH w e r e

reduced d e n a t u r e d

protein

the

second

approximately

protein

conditions.

and

the This

356

40 native

native

denat.

reduced

reduced O) a.

ß

20 f

2

Φ +•« Ο ν· α.

m

Ο

Fractions Fig.l:

m /

(0.2ml)

Determination of SH of Tryptophan 5-Monooxygenase

The e x p e r i m e n t was carried out as described under "Materials and Methods" and "Results". Chromatograms of t h e s e c o n d c o l u m n a r e s h o w n . Enzymes were: r e d u c e d / n a t i v e (left), reduced/denatured (middle) and non-reduced/native (right). Open bar: relative fluorescence, hatched bar: protein amount.

finding indicated that the DTT-treated enzyme is fully reduced with

respect

to

the

SH.

In

the

e n z y m e , about 50-60% of the total reagent. fully

of

SH w a s

the

non-reduced

labeled w i t h the

On the basis of the k n o w n n u m b e r of total

denatured

titration

case

by

and reduced

use

of

BSA,

which

SH of

was determined

5,5'-dithio-bis(2-nitrobenzoic

by

acid)

(DTNB), the n u m b e r of SH of the reduced tryptophan

5-mono-

o x y g e n a s e w a s calculated to be 0.67 mol per mol of

subunit

(MW. 5 0,000 ).

357 Discussion The sub-stoichiometric amount of SH per subunit of the reduced enzyme may be partially due to the different color yield in the protein assay based on BSA as the standard.

The differ-

ences in the quantum yield of the fluorescence of the probe (DACM) bound to different proteins were reported to be negligible under the full denaturation conditions (5).

Then the SH

number of 0.67 mol per mol of subunit calculated for the fully reduced enzyme indicated that the enzyme contains only one cysteine residue per subunit.

This result indicates one of

the features of tryptophan 5-monooxygenase, namely that the SH does not make intra-molecular(subunit) disulfide bond.

The

meaning of the sub-stoichiometric number of SH of the nonreduced enzyme, 0.36 mol per mol of subunit, was not clear, but a part of SH might have been masked by certain transient metal(s).

In this regard,

it is noteworthy that the apo-

enzyme prepared by treating

tryptophan

5-monooxygenase with

EDTA strictly required ferrous iron in the restoration of its activity (3).

If the dramatic activation by DTT is based on

the chelating activity of the mercaptide on non-effective metal(s) in addition to the activity as reductant of iron, an appropriate combination of a chelator and a reducing reagent should be able to activate the enzyme.

In the early works on

this line, the reconstitution of the apo-enzyme with ferrous iron in the absence of DTT was not reproducible nor quantitative; for example, less than 10% of the expected maximum activity was observed when the enzyme was simply mixed with ferrous iron before the assay.

But the enzyme showed near 70%

of its maximum activity when the apo-enzyme was mixed into a reaction mixture containing ferrous iron together with 200 mM ascorbate

and

2 mg/ml

of

catalase;

the

conditions

were

designed to minimize the presence of ferric iron even in the presence of oxygen (3).

Ascorbate itself did not support the

catalytic activity nor was capable of activating the enzyme. These results suggested that ferric iron is not only incapable

358 in supporting the enzyme's catalytic activity but also inhibitory to the reconstitution of the apo-enzyme with ferrous iron; the iron binding site(s) of the enzyme might be easily occupied by the non-effective species of iron, ferric iron. DTT selectively chelates ferric iron and reduces it to ferrous state.

Based on this model, the role of DTT in the activation

of tryptophan 5-monooxygenase by the anaerobic incubation can be explained

by assuming

that

the mercaptide

acted as a

chelator to depriving ineffective metals, presumably ferric iron, and allowed the enzyme to bind free ferrous iron, which was maintained in the reduced state only in the presence of the

mercaptide

or other

reducing

reagents

under

neutral

conditions.

References 1.

Ichiyama, Α., S. Hori, Y. Mashimo, T. Nukiwa, H. Makuuchi. 1974. FEBS lett. 40 , 88.

2.

Hasegawa, Η., M. Yanagisawa, F. Inoue, A. Ichiyama. 1983. In: Chemistry and Biology of Pteridines (J.A.Blair, ed.). Walter de Gruyter & Co., p.795.

3.

Hasegawa, H., A. Ichiyama. 1986. In: Chemistry and Biology of Pteridines (B.A. Cooper, V.M. Whitehead, eds. ). Walter de Gruyter & Co., p.36 9.

4.

Nakata, Η., Η. Fujisawa. 1 984. Eur.J.Biochem. 124 , 595.

5.

Yamamoto, Κ. , T. Sekine, Y. Kanaoka. 1 97 7. Anal .Biochem. 79. 8 3.

6.

Lowry, O.H., N.J. Rosebrough, 1951. J.Biol .Chem. 193 , 265.

A.L. Farr, R.J. Randall.

EFFECTS OF TETRAHYDROPTERINS ON DOPA PRODUCTION IN VITRO

AND

IN V I V O

M.

Bräutigam

Freie Universität Berlin, Institut für T h i e l a l l e e 6 9 - 7 3 , 1000 B e r l i n - 3 3 , F R G W.

Pharmakologie,

Pfleiderer

Universität Konstanz, 7750 K o n s t a n z , F R G

Fakultät für Chemie, Postfach

5560,

Introduction T h e r e p o r t of K e t t l e r ted

e t al.

6(R,S)-tetrahydrobiopterin

production

(1) t h a t i n t r a v e n t r i c u l a r l y (BH4)

accelerates

in rats p r o m t e d c o n s i d e r a b l e

striatal

interest

bility to i n c r e a s e a m i n e s y n t h e s i s in the C N S by replacement

in the

is l i m i t e d b y i t s p o o r o r a l

a n d e v e n m o r e by i t s p o o r e n t r y i n t o t h e b r a i n

(2). W e ,

(in g e n e r a l

lipophilic)

pterins

for t h e i r e f f i c a c y to a c c e l e r a t e

p r o d u c t i o n in d i f f e r e n t in v i t r o a n d in v i v o

possinatu-

availability

fore, tested v a r i o u s (H4-pterins)

more

DOPA

cofactor

therapy. The efficacy of a therapy w i t h the

r a l c o f a c t o r 6(R)-BH^

injec-

there-

tetrahydroDOPA

models.

Methods H^-pterins

where

sine h y d r o x y l a s e

s y n t h e s i s e d b y H. T r a u b (TH) a c t i v i t y

(prepared

(3). T e s t s o n from

p e r f o r m e d i n 5 m M T r i s H C l , p H 7.2 (4). A C r u d e fraction were

(P2) w a s p r e p a r e d f r o m r a t s t r i a t u m .

incubated

in a K r e b s - R i n g e r - p h o s p h a t e

DOPA a c c u m u l a t i o n w a s m e a s u r e d by HPLC w i t h

PC12

tyro-

cells)

were

synaptosomal Synaptosomes

buffer,

p H 7.2;

electrochemical

Unconjugated Pterins and Related Biogenic Amines © 1987 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

360

detect ion (ECD) after blockade of DOPA decarboxylase with 1 * 10"^ Μ 3-hydroxybenzyloxyamine dihydrogenphosphate (NSD 1055) (5). PC12 cells were cultured in Dulbecco's modification of Eagle's minimum essential medium; TH activity in intact PC12 cells was measured by DOPA accumulation after blocking DOPA decarboxylation (6). DOPA accumulation in rat striatum was determined after blocking DOPA decarboxylation with 3-hydroxybenzyl hydrazine (NSD 1015) (7). For determining tissue levels of 6(R)-BH4, 6-methyl-H4-pterin (6MPH4) and ethoxy-6-methylH4-pterin (Et-0-6MPH4) the H4-pterins were extracted in 0.2 Μ _ρ perchloric acid (containing 1 * 10 Μ dithioerythritol), separated by HPLC (ODS Hypersil, 5 jim; 20 mM citrate-phosphate buffer, pH 4.1, containing 1.5 mM octanesulfonic acid and 15% methanol) and detected by ECD (oxidation potential 300 mV).

Results Table 1 shows the activity of the various H4-pteridines as cofactors of TH in comparison to 6(R)-BH4. As can be noted, DOPA production (pmole/mg prot. * min) (6R)(6R,S (6R,S (6R,S (6R,S (6R,S (6R,S (6R,S (6R,S (6R,S (6R,S (6R,S

:etrahydrobiopterin -methylH 4 -pterin -hydroxymethylH 4 -pterin -methoxymethylH 4 -pterin -ethoxymethylH 4 -pterin -acetoxymethylH 4 -pterin -phenoxymethylH 4 -pterin -aminomethylH 4 -pterin -methylaminomethylH 4 -pterin -dimethylaminomethylH 4 -pterin -S-methylsulfonylmethyl- H 4 -pterin -ethoxymethyl-2,4-diamino--H 4 -pterindine

168. 0 79. 4 78. 0 1 86.6 271 . 4 1 48.0 68. 6 0. 0 1 .6 15. 6 31 .8 0. 0

5.9 1 .8 5.1 2.7 4.0 1 .3 + 1 .4

+ + + + + +

+ 1 .2 +

0.3

+ 1 .1

Table 1 : Effects of various H4-pterindines on TH activity. TH was prepared from PC12 cells; the concentration of the various H4-pteridines was 2 * 10 M.

361

especially the methoxy-e-methyl-H^-pterin (Me-O-öMPH^) and the Et-0-6MPH^ show a cofactor activity even higher than 6(R)BH4. To check the ability of the various H^-pterins to cross the cell membrane and to stimulate cellular DOPA production, PC12 cells were pretreated with 2,4-diamino-6-hydroxy-pyrimidine (DAO-Pyr) and were incubated with various H^-pterins. Pretreatment with DAO-Pyr inhibits de novo synthesis of 6{R)BH 4 and correspondingly cellular levels of 6(R)-BH4 and DOPA production are diminished (for details c.f. (8)). Figure 1 demonstrates that in particular Me-0-6MPH^ and Et-O-GMPH^ are more effective than 6(R)-BH4 to accelerate cellular DOPA production in the 6(R)-BH4 impoverished cells.

Acet-0-6MPH 4 Et-0-6MPH4 Me-0-6MPH4

>H

0H-6MPH 4 6MPH4 6(R)-BH4 controls

controls

I

1

1-

~Ί

"T" 40 20 30 DOPA production (pmol DOPA/mg protein · min) T

10

Fig. 1: Effect of various H^-pterins on DOPA production of PC12 cells. The cells were pretreated (24 h) with 1 * 10~2 Μ DAO-Pyr to yield a diminished endogenous intracellular 6(R)-BH4 content (8). In striatal synaptosomes (Fig. 2) the most significant effects on DOPA production were observed with Et-O-eMPH^. All compounds tested showed an inhibition of synaptosomal DOPA production at high concentrations. Despite its high effectivness in vitro no effects could be observed with high doses of Et-O6MPH4 on striatal DOPA production in vivo (Fig 3).

362 ΙΟ—·

8 -

Et-0-6MPH4

< Ο. §6" 6(R)-BH 4

ο Ε

a. Me-0-6MPH4 6ΜΡΗ4

4 c ο " υ •οσ 2