PURINE METABOLISM IN MAN-III Biochemical, Immunological, and Cancer Research

Edited by Aurelio Rapado Fundacion Jimenez Diaz Madrid, Spain R.W.E. Watts M.R.C. Clinical Research Centre Harrow, England

and Chris H.M.M. De Bruyn Department of Human Genetics University of Nijmegen Faculty of Medicine Nijmegen, The Netherlands

PLENUM PRESS · NEW YORK AND LONDON Contents of Part Β

I. PURINE METABOLISM PATHWAYS AND REGULATION

A. De Novo Synthesis; Precursors and Regulation

De Novo Purine Synthesis in Cultured Human Fibroblasts 1 R.B. Gordon, L. Thompson, L.A. Johnson, and B.T. Emmerson

Comparative Metabolism of a New Antileishmanial Agent, Allopurinol Riboside, in the Parasite and the Host Cell 7 D. J. Nelson, S.W. LaFon, G.B. Elion, J.J. Marr, and R.L. Berens

Purine Metabolism in Rat Skeletal Muscle 13 E. R. Tully and T.G. Sheehan

Alterations in Purine Metabolism in Cultured Fibroblasts with HGPRT Deficiency and with PRPPP Synthetase Superactivity 19 E. Zoref-Shani and 0. Sperling

Purine Metabolism in Cultured Endothelial Cells 25 S. Nees, A.L. Gerbes, B. Willershausen-Zönnchen, and E. Gerlach

Determinants of 5-Phosphoribosyl-l-Pyrophosphate (PRPP) Synthesis in Human Fibroblasts 31 K.0, Raivio, Ch. Lazar, H. Krumholz, and M.A. Becker

Xanthine Oxidoreductase Inhibition by NADH as a Regulatory Factor of Purine Metabolism 35 M.M. Jezewska and Z.W. Kaminski

vii viii CONTENTS OF PART Β

Β. Nucleotide Metabolism

Human Placental Adenosine Kinase: Purification and Characterization 41 CM. Andres, T.D. Palella, and I.H. Fox

Long-Term Effects of Ribose on Adenine Nucleotide Metabolism in Isoproterenol-Stimulated Hearts . . 45 H.-G. Zimmer, Η. Ibel, G. Steinkopff, and H. Koschine

C. Salvage Pathways

Purine Salvage Enzymes in Man and Leishmania donovani ... 51 T.A. Krenitsky, G.W. Koszalka, J.V. Tuttle, D. L. Adamczyk, G.B. Elion, and J.J. Marr

Regulation of Purine Salvage Enzymes in Έ. coli 57

R.A. Levine and M.W. Taylor

D. Catabolism

Purine Transport and the Cell Cycle 61 M.P. Rivera, M.R. Grau, J. Rigau, and A. Goday Hypoxanthine Transport in Human Erythrocytes 69 C. Salerno and Giacomello

Uptake of Adenosine in Human Erythrocytes 73 M. Kraupp, P. Chiba, and M.M. Müller

Effect of Actinomycin D on in vivo Purine Biosynthesis in Hamster Cells 79 M.W. Taylor, K.C. Gupta, and L. Zawistowich

Purine Catabolism in Isolated Hepatocytes: Influence of Coformycin 85 G. Van den Berghe, F. Bontemps, and H.G. Hers

II. ENZYMOLOGY AND PURINE METABOLISM

A. Phosphoribosyltransferases

Inactivation of Hyposanthine Guanine Phospho- ribosyltransferase by Guanosine Dialdehyde: An Active Site Directed Inhibitor 87 L.A, Johnson, R.B, Gordon, and B.T. Emmerson CONTENTS OF PART Β ix

Role of Human Hypoxanthine Guanine Phosphoribosyl- transferase in Nucleotide Interconversion .... 93 A. Giacomello and C. Salerno

Purification and Characterization of Mammalian Adenine Phosphoribosyltransferase 103 M.W. Taylor and H.V. Hershey

The Effect of Phosphoribosylpyrophosphate on Stability and Configuration of Hypoxan- thineguaninephosphoribosyltransferase and Adeninephosphoribosyltransferase from Human Erythrocytes Ill W. Gröbner and N. Zöllner

Chemical Modification of Hypoxanthine-phospho- ribosyltransferase and Its Protection by Substrates and Products 117 W.. Gutensohn and H. Jahn

Β.. Nucleoside Phosphoribosylating Enzymes

Structural Studies of Human Adenine Phosphoribosyl- transferase Purified by Affinity Chromatography 123 J,A. Holden, G.S. Meredith, and W.N. Kelley

Phosphoribosylpyrophosphate (PRPP) Synthetase Mutant in Salmonella typhimurium 131 B,. Jochimsen, Β. Garber, and J.S. Gots

C. Nucleoside Cleaving Enzymes

Methylmereaptopurine Ribonucleoside Toxicity in Human Fibroblasts: Inhibition of Phospho­ ribosylpyrophosphate Synthetase as well as Amidophosphoribosyltransferase 137 R..C.K. Yen and M.A. Becker

Adenosine Kinase: Regulation by Substrates, Magnesium, and pH 145 R..L. Miller and D.L. Adamczyk

Adenosine and Deoxyadenosine Kinase from Rat Liver .... 151 N.. Ogasawara, Y. Yamada, and H. Goto

D- Deaminating Enzymes

Radio immunochemical Analysis of Human Erythrocyte Adenosine Deaminase 157 P..E. Daddona, M.A. Frohman, and W.N. Kelley χ CONTENTS OF PART Β

Adenosine Deaminase Conversion Proteins: A Potential Role 163 P.P. Trotta and M.E. Balis

Regulatory Properties of AMP Deaminase Isozymes 169 N. Ogasawara, H. Goto, and Y. Yamada

Human Adenosine Deaminase: Stoichiometry of the Large Form Complex 177 P.E. Daddona and W.N. Kelley

Guanase from Human Liver - Purification and Characterization 183 R. Kuzmits, H. Stemberger, and M.M. Müller

Ε. Oxidating Enzymes

Nicotinamide and Liver Xanthine Oxidase 189 A. Di Stefano, Μ. Pizzichini, and Ε. Marinello

Xanthine Oxidase Activity in Human Intestines. Histochemical and Radiochemical Study 197 C. Auseher, Ν. Amory, P. van der Kemp, and F. Delbarre

III. PYRIMIDINE METABOLISM

Purine and Pyrimidine Metabolism in Hereditary Oroticaciduria During a 15-Year Follow-Up Study 203 D. R. Webster, H.A. Simmonds, C.F. Potter, and D.M.O. Becroft

Effect of Allopurinol on Pyrimidine Metabolism in Human White Blood Cells: Role of the Salvage Pathway 209 P. Banholzer, W. Gröbner, and N. Zöllner

Kinetics and Compartmentation of Erythrocyte Pyrimidine Metabolism 217 E. H. Harley, P. Zetler, and S. Neal

Simultaneous Determination of Rates of Purine and Pyrimidine Synthesis in Cultured Human Lymphoblasts and Fibroblasts 223 W.H. Huisman, K.O. Raivio, and M.A. Becker

Activities of Enzymes of Purine and Pyrimidine Metabolism in Nine Mycoplasma Species 231 M. Hamet, C. Bonissol, and P. Cartier CONTENTS OF PART Β x«

IV. LYMPHOCYTE PURINE METABOLISM RESEARCH

Increases in Purine Excretion and Rate of Synthesis by Drugs Inhibiting IMP Dehydrogenase or Adenylosuccinate Synthetase Activities 237 R.C. Willis and J.E. Seegmiller

Possible Role for 5T-Nucleotidase in Deoxyadenosine Selective Toxicity to Cultured Human Lymphoblasts 243 R.L. Wortmann, B.S. Mitchell, N.L. Edwards, and I.H. Fox

Cyclic Nucleotide Levels and Mechanism of Inhibition of Leucocyte Function by Adenosine Deaminase Inhibition 251 A.D. Meisel, Ch. Natarajan, G. Sterba, and H.S. Diamond

Purine Ribonucleoside and Deoxyribonucleoside Metabolism in Thymocytes 259 F.F. Snyder and T. Lukey

Molecular Mechanism(s) of Deoxyribonucleoside in T-Lymphoblasts 265 J.M. Wilson, B.S. Mitchell, and W.N. Kelley

Inhibition of Immune Cell Function by Adenosine: Biochemical Studies 271 T.P. Zimmerman, G. WoIberg, G.S. Duncan, R.D. Deeprose, and R.J. Harvey

Interactions Between Energy Metabolism and Adenine Nucleotide Metabolism in Human Lymphoblasts 277 S.S. Matsumoto, K.O. Raivio, R.C. Willis, and J.E. Seegmiller

Enzymes of Purine Interconversions in Subfractions of Lymphocytes 283 J.P.R.M. van Laarhoven, G.Th. Spierenburg, C.H.M.M. De Bruyn, and E.D.A.M. Schretlen

Measurement of the Rates of Synthesis and Degradation of Hypoxanthine-guanine phosphoribosyl- transferase in Human Lymphoblasts 289 P. Moore Mattes and W.N. Kelley xü CONTENTS OF PART Β

Human 51-Nucleotidase. Properties and Characterization of the Enzyme from Placenta. Lymphocytes and Lymphoblastoid Cells in Culture 295 W. Gutensohn

Metabolism and Toxicity of 9-Beta-D-Arabino- furanosyladenine in Human Malignant Τ Cells and Β Cells in Tissue Culture 299 D. A. Carson, J. Kaye, and J.E. Seegmiller

V. IMMUNE SYSTEM AND PURINE METABOLISM

Suppression of Cellular Immunity Due to Inhibition of Purine Nucleoside Phosphorylase by Allopurinol-Riboside 309 Y. Nishida, N. Kamatani, K. Tanimoto, and I. Akaoka

Lymphocyte 5T-Nucleotidase Deficiency: Clinical and Metabolic Characteristics of the Associated Hypogammaglobulinemia 315 N.L. Edwards, J.T. Cassidy, and I.H. Fox

Immunological Studies on Lesch-Nyhan Patients 321 C.H.M.M. de Bruyn, Ph. Gausset, J. Duchateau, E. Vamos, S. Kulakowski and G. Delespesse

Activity of Ecto-5T-Nucleotidase in Lymphoblastoid Cell Lines Derived from Carriers of Congenital X-Linked Agammaglobulinemia 327 L.F. Thompson, G.R. Boss, A. Bianchino, and J.E. Seegmiller

VI. CANCER RESEARCH

Adenosine Deaminase and Purine Nucleoside Phosphorylase in Acute and Chronic Lymphatic Leukemia 333 H. Ludwig, Η. Winterleitner, R. Kuzmits, and M.M. Müller

Purine Salvage Enzymes in Lymphocytes and Granulocytes from Patients with Small-Cell Carcinoma of the Lung 339 P. Nygaard and J. Mejer CONTENTS OF PART Β xiii

Treatment of Acute Lymphoblastic Leukemia with the Adenosine Deaminase Inhibitor 2r-Deoxycoformycin 347 B.S. Mitchell, Ch.A. Koller, and W.N. Kelley

Increase of Phosphoribosylpyrophosphate Levels in Cultured L1210 Leukemia Cells Exposed to Methotrexate 351 J.M. Buesa, A. Leyva, and H.M. Pinedo

Purine Salvage Pathway in Leukemic Cells 357 A. Goday, M.R. Grau, I. Jadraque, and M.P. Rivera

Biochemical Consequences of Treatment with the Adenosine Deaminase Inhibitor 2'-Deoxycoformycin 365 R.M. Paine, J.F. Smyth, and K.R. Harrap

In vitro and in vivo Effect of Deoxycoformycin in Human Τ Cell Leukemia 373 A.L. Yu, F.H. Kung, B. Bakay, and W.L. Nyhan

Uniqueness of Deoxyribonucleotide Metabolism in Human Malignant Τ Cell Lines 381 D.A. Carson, J. Kaye, S. Matsumoto, J.E. Seegmiller, and L. Thompson

VII. METHODOLOGY

High Performance Liquid Chromatography of Plasma Pyrimidines and Purines and Its Application in Cancer Chemotherapy 389 A. Leyva, J. Schornagel, and H.M. Pinedo

Urate-Binding Proteins in Plasma Studied by Affinity Chromatography 395 M.L. Ciompi, A. Lucacchini, D. Segnini, and M.R. Mazzoni

Chromatographic Determination of PRPP-Synthetase Activity in Human Blood Cells 401 P. Nygaard and K.F. Jensen

Purification of Myocardial Adenosine Kinase Using Affinity and Ion-Exchange Chromatography . . - . 409 M.P. Uitendaal, J.W. De Jong, E. Harmsen, and E. Keijzer xiv CONTENTS OF PART Β

Micromethods for the Measurement of Purine Enzymes in Lymphocytes 415 J.P.R.M. van Laarhoven, G.Th. Spierenburg, F.T.J.J. Oerlemans, and C.H.M.M. De Bruyn

A Rapid Screening Method for Inborn Errors of Purine and Pyrimidine Metabolism Using Isotachophoresis 421 H.A. Simmonds, A. Sahota, and R. Payne

Analysis of Serum Purines and Pyrimidines by Isotachophoresis 429 F. Oerlemans, Th. Verheggen, F. Mikkers, F. Everaerts, and C.H.M.M. De Bruyn

Determination of Uric Acid in Serum: Comparison of a Standard Enzymatic Method and Isotacho­ phoresis 435 F. Oerlemans, Th. Verheggen, F. Mikkers, F. Everaerts, and C.H.M.M. De Bruyn

Addition Products of Uric Acid and Formaldehyde 441 P.A. Simkin and Q.P. Lee

Automated Retrieval of Purine Literature 445 L. Ferreiro and A. Rey

Author Index 449

Subject Index 453 Contents of Part A

I. CLINICAL GOUT

The Natural History of Hyperuricemia Among Asymptotic Relatives of Patients With Gout . 1 T.-F. Yii and C. Kaung

The Clinical Differentiation of Primary Gout From Primary Renal Disease in Patients With Both Gout and Renal Disease 9 Β. T. Emmerson, P. J. Stride and G. Williams

Renal Failure in Young Subjects With Familial Gout 15 H. A. Simmonds, J. S. Cameron, C. F. Potter, D. Warren, T. Gibson, and D. Farebrother

Family Study of Lipid and Purine Levels in Gout Patients and Analysis of Mortality 21 L. G. Darlington, J. Slack, and J. T. Scott

Uric Acid Turnover in Normals, in Gout and in Chronic Renal Failure Using 14C-Uric Acid 27 C. Vitali, G. Pasero, A, Clerico, L. Riente, N. Molea, A. Pilo, G. Mariani, and R. Bianchi

Erythrocyte Adenosine-Deaminase Activity in Gout and Hyperuricemia 33 A. Carcassi, P. Macri, G. Chiaroni, and S. Boschi

Clinical Variability of the Gouty Diathesis 39 W. J. Arnold and R. A. Simmons

xv xvi CONTENTS OF PART A

Clinical Features of 4,000 Gouty Subjects in Japan 47 N. Nishioka and K. Mikanagi

Frequency of Chondrocalcinosis of the Knees and Avascular Necrosis of the Femoral Heads in Gout, a Controlled Study 55 A. Stockman, L. G. Darlington, and J. T. Scott

Gaschromatographic Evaluation of Urinary 17-Ketosteroids, Etiocholanolone and Dehydroepiandrosterone in Primary Gout and Hyperuricemia 59 A. Carcassi, F. Lore, G. Manasse, P. Macri, and M. Pisano

Hormonal Aspects of Gouty Patients 65 U. Valentini, G. Riario-Sforza, R. Marcolongo, and E. Marinello

Determination of Tubular Secretion of Urate in Healthy and Gouty Men 73 L. B. Sorensen and D. J. Levinson

Ribose Tolerance in Gouty Patients 81 M. Pizzichini, R. Marcolongo, and E. Marinello

II. URIC ACID AND RENAL STONES

Urinary Urate and Uric Acid Relative Saturation in Normouricuric Calcium Oxalate Stone Formers With Normal Urinary Calcium Oxalate Saturation 87 M. Labeeuw, C. Gerbaulet, N. Pozet, P. Zech, and J. Traeger

Correlation Between the Uric Acid and Calcium Concentration in Urine. Results of a Long Term Study on Recurrent Stone-Formers and Healthy Controls 93 P. Leskovar, R. Härtung, and M. Kratzer

Interaction of Hyperuricuria and Hyperoxaluria on Renal Calcium Oxalate Stone Formation 99 F. Hering, Κ.-Η. Bigalke, and W. Lutzeyer

Uric Acid/Calcium Oxalate Nephrolithiasis. Clinical and Biochemical Findings in 86 Patients 109 A. Rapado, J.M. Castrillo, M. Diaz-Curiel, M. L. Traba, M. Santos, L. Cifuentes-Delatte CONTENTS OF PART Α ^,.

The Uric Acid: Cystine Correlation in the Urine of Recurrent Calcium Oxalate Stone-Formers and Healthy Controls 115 P. Leskovar, R. Härtung, and M. Kratzer

The Role of Urate in Idiopathic Calcium Urolithiasis 121 S. R. Silcock

Mineralogie Composition of 66 Mixed Urinary Calculi of Calcium Oxalate and Uric Acid 129 J. R. Minon-Cifuentes, M. Santos, and L. Cifuentes-Delatte

Hyperuricemia and Cystinuria 135 F. Linari, M. Marangella, B. Malfi, G. Vacha, Μ. Bruno, G. Giorcelli, and B. Fruttero

Monosodium Urate Monohydrate as Spherulites 141 J. J. Fiechtner and P. A. Simkin

III. CLINICAL AND PHYSIOLOGICAL ASPECTS OF PURINE METABOLISM

Tumoural Hypouricemia 145 A. Lesmes, M. Diaz-Curiel, and J. M. Castrillo

Hereditary Renal Hypouricemia With Hyperuricosuria and Variably Absorptive Hypercalciuria and Urolithiasis - A New Syndrome 149 0. Sperling and A. de Vries

Hereditary and Environmental Factors Influencing on the Serum Uric Acid Throughout Ten Years Population Study in Japan 155 K. Nishioka and K. Mikanagi

The Natural History of Urate Overproduction in Sickle Cell Anemia 161 H. S. Diamond, A. D. Meisel, and D. Holden

Salvage Pathway in Erythrocytes of Patients With Psoriasis 167 G. Partsch, F. Mayer, R. Eberl, and A. Luger

Serum 5-Nucleotidase in Progressive Muscular Dystrophy 173 F. Lähoda and K. Baier CONTENTS OF PART A xviü

Purine Metabolism in Duchenne Muscular Dystrophy 177 C. Η. Μ. M. De Bruyn, S. Kulakowski, C. A. van Bennekom, P. Renoirte, and Μ. M. Müller

Metabolism of Adenine and Adenosine in Erythrocytes of Patients with Myotonic Muscular Dystrophy (MMD) 183 Μ. M. Müller, Μ. Frass, and B. Mamoli

Clinical and Enzymological Studies in a Child With Type I Glycogen Storage Disease Associated With Partial Deficiency of Hepatic Glucose- 6-Phosphatase 189 G. Nuki and J. Parker

Differential Absorption of Purine Nucleotides, Nucleosides and Bases 203 C. F. Potter, A. Cadenhead, H. A. Simmonds, and J. S. Cameron

Influence of Dietary Protein on Serum and Urinary Uric Acid 209 W. Löffler, W. Gröbner, and N. Zöllner

Effect of Hypoxanthine in Meat on Serum Uric Acid and Urinary Uric Acid Excretion 215 W. K. Spann, W. Gröbner, and N. Zöllner

On the Mechanism of the Paradoxical Effect of Salicylate on Urate Excretion 221 H. S. Diamond, G. Sterba, K. Jayadeven, and A. D. Meisel

The Uricosuric Action of Protein in Man 227 F. Matzkies, G. Berg, and H. Mädl

Hypoxic Effects on Purine Metabolism Studied With High Pressure Liquid Chromatography 233 R. A. Harkness, R. J. Simmonds, and M. C. O'Connor

Urate Metabolism in a Mongrel Dog 237 P. A. Simkin COINTENTS OF PART A xix

IV. STUDIES ON XANTHINURIA

Thie Effect of Weight Reduction on Plasma and Urinary Levels of Oxypurines in an Obese Xanthinuric Patient 241 C. Auscher, C. Pasquier, N. Amory, G. Gay, A. Aisene, and G. Debry

Xamthinuria: The Cause of Hypouricemia in Hepatic Disease 247 H. J. Castro-Mendoza, A. Rapado, C. De La Piedra, and J. M. Castrillo

Xainthine-Coproporphyrin III 251 H. J. Castro-Mendoza

V. THERAPEUTICAL ASPECTS OF DISORDERS IN PURINE METABOLISM

A Controlled Study of the Effect of Long Term Allopurinol Treatment on Renal Function in Gout 257 T. Gibson, H. A. Simmonds, C. Potter, and V. Rogers

St:udies With Allopurinol in Patients With Impaired Renal Function 263 G. B. Elion, F. M. Benezra, T. D. Beardmore, and W. N. Kelley

Phiarmacological Effects of 1,3,5-Triazines and Their Excretion Characteristics in the Rat 269 M. Hropot, F. Sörgel, Β. v. Kerekjärto, H. J. Lang, and R. Muschaweck

Tiienilic Acid in the Treatment of Gout and Hypertension 277 T. Gibson, C. Potter, H. A. Simmonds, V. Rogers, and R. I. Gleadle

Benzbromarone as a Long-Term Uricosuric Agent 283 R. Bluestone, J. Klinenberg, and I. K. Lee

Thie Action of Benzbromarone in Relation to Age, Sex and Accompanying Diseases 287 H. Ferber, U. Bader, and F. Matzkies

Covalitin®: A New Drug for the Treatment of Uric Lithiasis 295 T. Covaliu XX CONTENTS OF PART A

VI. MUTATIONS AFFECTING PURINE METABOLISM

A. Phosphoribosyltransferases

Hypoxanthine Salvage in Man: Its Importance in Urate Overproduction in the Lesch-Nyhan Syndrome 301 N. L. Edwards, D. P. Recker, and I. H. Fox

Aspect of Purine Metabolic Aberration Associated With Uric Acid Overproduction and Gout 307 L. C. Yip, T.-F. Yü, and Μ. E. Balis

Properties of a Mutant Hypoxanthine-Phosphoribosyl- transferase in a Patient With Gout 313 W. Gröbner and W. Gutensohn

Variation in Human HPRT and Its Relationship to Neurologic and Behavioral Manifestations 317 B. Bakay, E. Nissinen, L. Sweetman, U. Franeke, and W. L. Nyhan

High HPRT Activity in Fibroblasts from Patients With Lesch-Nyhan Syndrome due to Bacterial "L-Form" Contamination 327 I. Willers, S, Singh, K. R. Held, and H. W. Goedde

Kinetics of a HGPRT Mutant Showing Substrate Inhibition 333 Ε. H. Harley, C. M. Adnams, and L. M. Steyn

B. Nucleoside Phosphoribosylating Enzymes

Spectrum of 2,8-Dihydroxyadenine Urolithiasis in Complete APRT Deficiency 337 H. A. Simmonds, T. M. Barratt, D. R. Webster, A. Sahota, K. J. Van Acker, J. S. Cameron, and M. Dillon

Complete Adenine Phosphoribosyltransferase (APRT) Deficiency in Two Siblings: Report of a New Case 343 P. Cartier, M. Hamet, A. Vincens, J. L. Perignon

Inheritance of Adenine Phosphoribosyltransferase (APRT) Deficiency 349 K. J. Van Acker, H. A. Simmonds, C. F. Potter, and A. Sahota CCHNTENTS OF PART A xxi

Iimmunological Evaluation of a Family Deficient in Adenine Phosphoribosyl Transferase (APRT) 355 W. J. Stevens, Μ. E. Peetermans, and K. J. Van Acker

Activities of Amidophosphoribosyltransferase and Purine Phosphoribosyltransferases in Developing Rat Brain 361 J. Allsop and R. W. E. Watts

Puirine Nucleoside Phosphorylase Deficiency; Genetic Studies in a Dutch Family 367 G. E. J. Staal, M. J. M. van der Vlist, R. Geerdink, J. M. Jansen-Schillhorn van Veen, B. J. M. Zegers, and J. W. Stoop

C. Nucleoside Cleaving Enzymes

Abnormal Regulation of Purine Metabolism in a Cultured Mouse T-Cell Lymphoma Mutant Partially Deficient in Adenylosuccinate Synthetase 375 B. Ullman, M. A. Wormsted, B. B. Levinson, L. J. Gudas, A. Cohen, S. M. Clift, and D. W. Martin, Jr.

Suiperactive Phosphoribosylpyrophosphate Synthetase With Altered Regulatory and Catalytic Properties 387 M. A. Becker, K. 0. Raivio, B. Bakay, W. B. Adams, and W. L. Nyhan

AM1P Phosphatase Activity in Human Term Placenta: Studies on Placental 5f-Nucleotidase 393 Μ. H. Maguire and T. P. Krishnakantha

D. Deaminating Enzymes

Adlenosine and Deoxyadenosine Metabolism in the Erythrocytes of a Patient With Adenosine Deaminase Deficiency 397 A. Sahota, H. A. Simmonds, C. F. Potter, J. G. Watson, K. Hugh-Jones, and D. Perrett

Complete Adenosine Deaminase (ADA) Deficiency Without Immunodeficiency, and Primary Hyperoxaluria, in a 12-Year-01d Boy 403 J. L. Perignon, M. Hamet, P. Cartier, and C. Griscelli xxii CONTENTS OF PART A

Metabolism of Adenosine and Deoxyadenosine by Stored Human Red Cells 409 G. R. Bartlett

Adenosine Deaminase and Purine Nucleoside Phosphorylase Activities During Culturing of Fibroblasts 415 M. P. Uitendaal, F. T. J. J. Oerlemans, C. Η. Μ. M. De Bruyn, T. L. Oei, and P. Hösli

S-Adenosylhomocysteine Metabolism in Adenosine Deaminase Deficient Cells 421 M. S. Hershfield and Ν. M. Kredich

Altered Deoxynucleoside Triphosphate Levels Paralleling Deoxyadenosine Toxicity in Adenosine Deaminase Inhibited Human Lymphocytes 427 H. G. Bluestein, L. F. Thompson, D. A. Albert, and J. E. Seegmiller

Author Index 433

Subject Index 437 PURINE METABOLISM IN CULTURED CORONARY ENDOTHELIAL CELLS

S. Nees, A.L. Gerbes, B. Willershausen-Zönnchen and E. Gerlach

Physiologisches Institut der Universität München

Pettenkoferstr. 12, D-8000 München 2

Although it is well known that endothelial cells are involved in several biological processes such as transport*!, hemostasis^, synthesis of collagen-^, histamine^ and prostaglandins^, our know• ledge concerning intermediary metabolism of the endothelium is ra• ther limited. In the course of studies on interrelationships bet• ween heart function and cardiac metabolism^7 we became interested in some features of purine metabolism of coronary endothelial cells. Our interest was initiated by the assumption that these cells might contribute to the production of vasoactive adenosine which is considered to play an important role in the metabolic re• gulation of coronary blood flow^»^. The studies - not possible of course to be performed under in vivo conditions - were carried out on cultured endothelial cells isolated from coronary vessels of guinea pig hearts as recently described^.

MATERIALS AND METHODS

Culture Medium 199 (Seromed, München) containing penicilline (200 TJ/ml) and streptomycine (200 ug/ml) was supplemented with fe• tal calf serum (20%) and L-glutamine (2 mM). Column packings (to• tally porous silica) for High Pressure Liquid Chromatography (HPLC) were obtained from Macherey & Nagel, Düren. Nucleotides, nucleo• sides and bases for calibration were purchased from Boehringer Mannheim, all other materials of highest available purity from Merck, Darmstadt.

Preparation of cells and cell culture: Guinea pig hearts were cannulated through the aorta, and their coronary system, washed

25 26 S. NEES ET AL. free of blood, was filled with an isotonic buffer solution con­ taining collagenase and trypsin (0.1% each). After an exposure of 20 min perfusion was started again and all endothelial cells which had been detached, were collected from the perfusate by centrifu- gation. Subsequently, the cells were washed with culture medium and seeded in culture dishes. Cultivation was performed at 37°C in a humidified air atmosphere containing 3% CO2. Depending on the inoculum confluency was reached after 2 to 4 weeks. Contaminations with fibroblasts and smooth muscle cells were usually less than 2% As judged by electron microscopy the cultivated endothelial cells revealed important morphological criteria of endothelial cells in vivo (clusters of free ribosomes, smooth and rough endoplasmic re­ ticulum, clumps of coarse filaments, fine filaments and prominent microtubules in the cytoplasma).

Analysis of nucleotides, nucleosides and bases: Cultured en­ dothelial cells were extracted with 0.4 Ν perchloric acid. Quanti­ tation of the different purine compounds in the neutralized cell extracts was carried out by application of specially elaborated HPLC-techniques using weak anion exchange columns for the separa­ tion of the nucleotides and reverse phase columns for nucleosides and bases.

Determination of enzyme activities: Specific activities of enzymes involved in nucleotide metabolism were measured in a 20 000 g membrane preparation as well as in a soluble 200 000 g supernatant fraction of endothelial cells. Enzyme tests were per­ formed using standard procedures, substrates and products were se­ parated by HPLC.

RESULTS AND DISCUSSION

In Table 1 mean values from three individual series of analy­ ses concerning contents of purine nucleotides, nucleosides and ba­ ses in non-growing confluent endothelial cell cultures are listed. For reasons of comparison respective data for normoxic myocardial tissue are also given. Obviously, endothelial cells contain extra­ ordinarily high amounts of ATP, ADP and AMP. The sum of the adenine nucleotides (£?ATP, ADP, AMP) reaches with more than 15 umoles/g a value which is about three times higher than the mean adenine nucleotide content of cardiac tissue. Another interesting feature of endothelial cells concerns their high levels of adenine nucleo­ tide degradatives. The contents of adenosine, inosine, adenine and hypoxanthine are about 1 to 2 orders of magnitude higher than the respective values for the myocardium. In contrast to the high le­ vels of adenine nucleotides guanine nucleotides are present in en­ dothelial cells only in small quantities, which are similar to those in myocardial and other tissues. PURINE METABOLISM IN CULTURED ENDOTHELIAL CELLS 27

Table 1: Content of adenine nucleotides and their dephosphorylated degradatives in confluent coronary endothelial cells and in myocar• dial tissue of guinea pigs. Mean values from three individual series of analyses of 11 culture dishes each.

Endothelial cells Myocardium nmoles/g nmoles/g

ATP 11 960 4 280 ADP 2 760 1 050 AMP 630 160

^> fATP, ADP, AMP 1 5 350 5 490

Adenosine 87 2 Inosine 100 1 .2 Adenine 60 0.5 Hypoxanthine 50 0.9

GTP 233 200 GDP 157 100 GMP 37 23 Guanosine *) *) Guanine *) *)

not detectable

Additional experiments revealed that growth state of the cul• tures did not profoundly influence the total content of adenine nucleotides. Furthermore, incubation of confluent cell cultures in purine-free media for three days did not result in any detectable reduction of the adenine nucleotide content. On the other hand, endothelial cells proved to be sensitive to lack of oxygen. It is evident from the data in Fig. 1 that brief periods of anoxic incu• bation (1.5 and 3 min, respectively) cause a pronounced decrease of ATP with a corresponding increase in ADP and AMP levels. Simul• taneously, remarkable amounts of adenosine are formed and released from the cells into the incubation medium.

It appears from all these observations that the extremely high adenine nucleotide levels are very likely a specific feature of cultured endothelial cells. This view is further supported by de• terminations of activity values of enzymes involved in degradation and synthesis of adenine nucleotides (Table 2). While 51-nucleoti• dase activity in endothelial cells exceeds by far that of myocar- 28 S. NEES ET AL

NORMOXIA ANOXIA

1.5min 3min 10

l nmoles/g ]

Fig. 1: Influence of anoxia on levels of adenine nucleotides in co• ronary endothelial cells and on the release of adenosine into the medium (mean values from 3 experiments)

dial tissue, the opposite holds true for adenosine deaminase, the activity of which is much higher in the myocardium. These differen• ces in the pattern of enzyme activities may reasonably explain that endothelial cells contain adenosine in rather high amounts compared with the small quantities of this nucleoside found in the myocardium. As is further evident from the data in Table 2, acti• vities of G-6-PDH and PRPP-synthetase, indirectly involved in the biosynthesis of nucleotides, proved to be much higher in endotheli• al cells than in cardiac tissue. These findings are in accordance with results from preliminary studies, in which by use of 1-^C- glycine and ^C-labeled purine bases purine nucleotide synthesis in endothelial cells was shown to proceed via salvage and de novo pathways.

SUMMARY

Endothelial cells from coronary vessels of guinea pig hearts were isolated, cultivated and morphologically characterized.- Cells PURINE METABOLISM IN CULTURED ENDOTHELIAL CELLS 29

Table 2: Enzyme activities in cultured coronary endothelial cells and in cardiac tissue from guinea pigs.

Specific activity [nmoles/min *mg]

Endothelial Cardiac cells tissue

5'-Nucleotidase [E.C. 3.1.3.5] 95 13.6

Alkaline phosphatase [E.C. 3.1.3.1] 14.6 24

AMP deaminase [E.C.3.5.4.6] 1 .1 2.2

Adenosine deaminase [E.C. 3.5.4.4] 3.4 28

Glu-6-P-dehydrogenase [E.C. 1.1.1.49] 12.7 4.2

PRPP-synthetase [E.C. 2.7.6.6] 6.58 1 .9

APR-transferase [E.C. 2.4.2.7] 0.7 0.2

GPR-transferase [E.C. 2.4.2.8] 0.3 0.1

Adenylate cyclase [E.C. 4.6.1.1] 0.1 0.4

Phosphodiesterase [E.C. 3.1.4.1] 2.1 15

from confluent cultures contained adenine nucleotides and their de- phosphorylated degradatives in exceptionally high amounts.- Adenine nucleotide levels were only slightly influenced by the growth state of the cultures and remained stable during incubation for three days in purine-free medium. In contrast, brief incubation of endo• thelial cells under anoxic conditions resulted in a substantial breakdown of adenine nucleotides associated with an enhanced for• mation and release of adenosine.- Measurements of specific activi- 30 S. NEES ET AL ties of enzymes involved in adenine nucleotide synthesis and de­ gradation lend additional support to the view that a very active adenine nucleotide metabolism is a typical feature of cultured coronary endothelial cells.

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