Other Pergamon publications of related interest Books MILDNER et al Trends in Enzymology TORCHINSKY Sulfur in Proteins Review Journal Molecular Aspects of Medicine A m i ne Oxidases in Clinical Research V. Ć. GORKIN Institute of Biological and Medical Chemistry Academy of Medical Sciences Pogodinskaya Street 10 Moscow 119121 USSR Translation editor J. HICKLIN PERGAMON PRESS OXFORD NEW YORK TORONTO SYDNEY PARIS FRANKFURT U.K. Pergamon Press Ltd., Headington Hill Hall, Oxford 0X3 OBW, England U.S.A. Pergamon Press Inc., Maxwell House, Fairview Park, Elmsford, New York 10523, U.S.A. CANADA Pergamon Press Canada Ltd., Suite 104, 150 Consumers Rd., Willowdale, Ontario M2J 1P9, Canada AUSTRALIA Pergamon Press (Aust.) Pty. Ltd., P.O. Box 544, Potts Point, N.S.W. 2011, Australia FRANCE Pergamon Press SARL, 24 rue des Ecoles, 75240 Paris, Cedex 05, France FEDERAL REPUBLIC Pergamon Press GmbH, 6242 Kronberg-Taunus, OF GERMANY Hammerweg 6, Federal Republic of Germany Translation copyright © 1983 Pergamon Press Ltd. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic tape, mechanical, photocopying, recording or otherwise, without permission in writing from the publishers. First English edition 1983 Library of Congress Cataloging in Publication Data Gorkin, V. Z. Amine oxidases in clinical research. Includes bibliographical references and index. 1. Amine oxidase. I. Title. [DNLM: 1. Amine oxidoreductases. QU 140 G669a] QP603.M6G67 1983 612\01518 82-18132 ISBNO-08-025523-X In order to make this volume available as economically and as rapidly as possible the author 's typescript has been reproduced in its original form. This method unfortunately has its typographical limitations but it is hoped that they in no way distract the reader. Printed in Great Britain by A. Wheaton & Co. Ltd., Exeter Foreword Abnormalities of key enzyme systems determine the most important features of the metabolic disturbances that occur in pathological states, and research on these enzyme systems is of paramount importance to enable us to answer a number of fundamental questions in biological and medical chemistry which are of direct relevance to current trends in theoretical and clinical medicine. Amine oxidases, which catalyse the deamination of biogenic amines and neuro­ transmitters, also play a part in regulating cardiovascular functions, and the growth and division of cells. The amine oxidases comprise the membrane- bound flavin-containing monoamine oxidases, which according to current opinion are also involved in regulating the permeability of biomembranes, and the copper-containing diamine oxidases, polyamine oxidases and lysyl oxidases, which catalyse the deamination of epsilon-amino groups of lysine residues during the formation of cross-links in the connective-tissue proteins collagen and elastin. Although the amine oxidases were discovered many years ago, the relevance of the functions that have been postulated for them in medical and biological problems has in recent years produced a rapid development of research inter­ est. Amine oxidase activity can be inhibited to a variable extent in the human body. The compounds which have this effect (the so-called monoamine oxidase inhibitors) are used in clinical medicine as drugs. Irrespective of current opinion regarding the therapeutic efficacy of monoamine oxidase inhibitors, it is evident that amine oxidases, which are inhibited by these drugs, do carry out key functions in the metabolism of biogenic amines, and possibly of other nitrogenous compounds under normal and pathological conditions. Data that are gradually accumulating are demonstrating the significance of abnormal catalytic activity of the amine oxidases, the patho­ genesis of a number of diseases of the central nervous and cardiovascular systems, and are proving the diagnostic value of estimating amine oxidase activity in clinical medicine. The Institute of Biological and Medical Chemistry in Moscow, which is a branch of the Academy of Medical Sciences of the USSR, has been conducting studies on the metabolism of biogenic amines and other nitrogenous compounds and research on amine oxidases for many years. The results of our studies have been presented in a number of papers and monographs (for example, A.E. Braunstein, Biochemistry of Amino Acid Metabolism, 1949; multi-author ν vi Amine Oxidases: Clinical Aspects monographs edited by V.N. Orekhovich, Chemical Bases of Biological Processes, 1962; The Molecular Bases of Pathology, 1966; Chemical Mechanisms of Regulating the Activity and Biosynthesis of Enzymes, 1969), one of which is this present work by V.Z. Gorkin. This monograph summarizes our research on the isolation and purification of membrane-bound enzymes (as exemplified by mitochondrial monoamine oxidases), the investigation of their catalytic properties and the regulation of amine oxidase activity. Of fundamental importance is the discovery by Gorkin and his coworkers of regulation of amine oxidase activity via a post-translational mechanism of reversible qualitative modification, or transformation. In these circumstances amine oxidase activity is accompanied by partial oxidation of the -SH groups in proteins and/or by an increase in the rate of hydrolysis of peptide bonds in tissue proteins and peptides. This way of altering amine oxidase activity appears to be especially important in certain pathological conditions and has to be considered when evaluating the possible biological roles of the amine oxidases and the pathogenetic significance of abnormal catalytic activity. Further studies on the nature of amine oxidases and their inhibitors and activators are promising lines of research in a number of basic problems of biological and medical chemistry. As an analytical summary of current knowledge, this book will be of great use to investigators about to embark on research in this important and fascinating field. V.N. Orekhovich, July, 1979 Director, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow 1 Introduction Many physiologically occurring amines have long been known to possess biological activity. Mechnikov (1913) was impressed by the striking pharm­ acological effect of biogenic (synthesized from biological sources) amines and considered these compounds important in human physiology and pathology. Recent studies have tended to focus on phenylalkylamines ($-phenylethylamine, tyramine, octopamine, dopamine, noradrenaline, adrenaline), indolealkylamines (tryptamine, 5-hydroxytryptamine, maxamine), imidazolealkylamines (histamine), diamines (putrescine, cadaverine), polyamines (spermine, spermidine) and many other low-molecular nitrogenous compounds synthesized in the course of investigations into amino acid metabolism (Braunstein, 1949) and character­ ized by high biological activity. Amine oxidases catalysing oxidative deamination are the key enzymes in the metabolism of these compounds. R-<:H-NH + 0 + H 0 •> R-CHO + NH + H 0 2 2 2 2 3 2 2 (R— represents a fatty-aromatic or aliphatic residue). In accordance with the International Enzyme Nomenclature (1972), amine oxi­ dases belong to class 1 (oxidoreductases), within which a subclass 1.4 (oxidoreductases acting on the 0Η-ΝΗ group of doners) is distinguished. 2 This subclass can further be subdivided into subgroup 1.4.3. EC 1.4.3.4. Recommended name: amine oxidase (flavin-containing). Alternative names: monoamine oxidase, tyramine oxidase, tyr- aminase, amine oxidase, monoamine 0 transhydrogenase, adren­ 2 aline oxidase. Systematic name: amine:oxygen oxidoreductase (deaminating)(flavin-containing). These flavoproteins act on primary, secondary and tertiary amines. EC 1.4.3.5. Recommended name: pyridoxaminephosphate oxidase. Systematic name: pyridoxamine phosphate:oxygen oxidoreductase (deaminating), This flavoprotein also oxidises pyridoxine 5-phosphate and pyridoxine. EC 1.4.3.6. Recommended name: amine oxidase (pyridoxal-containing). Other names: diamine oxidase, diamine oxyhydrase, diamino0 trans- 2 AOCR - A* 1 2 Amine Oxidases: Clinical Aspects hydrogenase, histaminase. Systematic name: amine:oxygen oxidoreductase (deaminating)(pyridoxal-containing). These enzymes, which are considered as pyridoxal-phosphate-dependent, copper-containing proteins, oxidize primary monoamines (includ­ ing histamine) and diamines. EC 1.4.3.8. Recommended name: ethanolamine oxidase. Systematic name: ethanolamine:oxygen oxidoreductase (deaminating). This cobamide- dependent enzyme catalyses the oxidative deamination of ethanol­ amine with the formation of glycoaldehyde. EC 1.4.3.9. Recommended name: tyramine oxidase. Systematic name: tyramine: oxygen oxidoreductase (deaminating). This FAD-containing enzyme also acts on dopamine and slowly oxidizes secondary amines. The present book considers primarily those enzymes denoted as EC 1.4.3.4 (flavin amine oxidases, or monoamine oxidases) and EC 1.4.3.6 (copper-con­ taining amine oxidases, or diamine oxidases). Some amine oxidases (e.g. benzylamine oxidases, which occur in mammlian blood and tissues) and lysyl oxidases, found in connective tissue, are also discussed, although these enzymes are still not included in the International Enzyme Nomenclature. The properties of these enzymes are quite distinct from those of either monoamine oxidases or diamine oxidases, but studies on them may have impor­ tant clinical implications. Nonspecific amine oxidases (e.g. caeruloplasmin and the microsomal amine oxidase of liver tissue) are not considered in this book. The first data on the existence of monoamine oxidase were published by Hare (1928). Histaminase (diamine oxidase) was discovered by Best (1929). The earliest studies on mammalian serum amine oxidases were published by Hirsch (1953) (Blaschko, 1974; Kapeller-Adler, 1970). Lysyl oxidases, which deaminate peptide-bound lysine residues during the synthesis of desmosine, isodesmosine and lysinonorleucine in connective tissue proteins, were dis­ covered only in the late 1960s (Pinell and Martin, 1968). Lysyl oxidases are related to diamine oxidases and serum amine oxidases. In the late 1930s, it was postulated that the deamination of tyramine, nor­ adrenaline, adrenaline and aliphatic monoamines was catalysed by a single enzyme, denoted monoamine oxidase, and that oxidation of histamine and ali­ phatic diamines with a short carbohydrate chain in the molecule, putrescine (1,4-tetramethylene diamine) or cadaverine (1,5-pentamethylene diamine) is catalysed by another enzyme, diamine oxidase (Zeller, 1938). Monoamine oxidase inhibitors were discovered in the early 1950s (Zeller and Barsky, 1952). These compounds block monoamine oxidase activity and have been used therapeutically to treat depression and cardiac insufficiency. All the known monoamine oxidase inhibitors have characteristic disadvantages; nevertheless the development of potent selective inhibitors is considered to be an important goal in the design of new drugs (Gorkin, 1977b; Knoll and coworkers, 1978). The monoamine oxidase inhibitors made it possible to establish the fact that biogenic amines are synthesized not only in the gastrointestinal tract by the activity of microorganisms, but in all human tissues. However, free biogenic amines are rapidly deaminated by amine oxidases — the key enzymes of biogenic amine metabolism — which prevent an excess from accumulating in the tissues (Hess and coworkers, 1959). This discovery was important in the development of modern ideas concerning the significance of abnormal metabolism of biogenic amines in the pathogenesis of diseases of the cardiovascular and nervous systems, and for helping to elucidate the mode of action of a number of psychotropic and hypotensive drugs. Introduction 3 The number of publications devoted to amine oxidases began to increase dram­ atically from the late 1950s. Some of the published data seemed to be contra­ dictory, but the apparent discrepancies were often the result of using different amines as substrates when estimating amine oxidase activity. It gradually became apparent that the enzyme denoted by the term monoamine oxidase is not a single macromolecule with an active site characterized by broad substrate specificity (i.e. deaminating any monoamine), but a family of related enzymes (or catalytic sites on the surface of a single macro- molecule) , each with a sharper substrate specificity (Gorkin, 1963). This concept was supported by data on the separation of various monoamine oxi­ dase subfractions (Gorkin, 1963; Moskvitina and coworkers, 1979), and by studies on selective inhibition of the deamination of various substrates by monoamine oxidase inhibitors (Gorkin and coworkers, 1964a). This second approach developed rapidly after the discovery of clorgyline (Johnston, 1968) and deprenyl (Knoll and coworkers, 1965, 1978; Knoll and Magyar, 1972), potent selective inhibitors of this type. The current view (Fowler and coworkers, 1978) is that at least two main types of monoamine oxidases may be identified in human and animal tissues by means of inhibitor analysis. Type A monoamine oxidase is defined as the enzyme component inhibited by low (0.1 uM) concentrations of clorgyline. The important neurotransmitters, noradrenaline and 5-hydroxytryptamine (sero­ tonin) were found to be specific substrates. The activity of the type Β enzyme is inhibited by much higher concentrations of clorgyline. Deprenyl is a selective inhibitor of this form, some specific substrates being benzyl- amine, ß-phenylethylamine and N-methylhistamine. Many important amines (for example, tyramine) are deaminated by both forms. The molecular basis of the difference between the two forms is still unresolved (Cawthon and Breakefield, 1979). Purified preparations of monoamine oxidases contain a flavin component (FAD), covalently bound with the protein component of these enzymes, and SH-groups. The role of metal ions in the action of monoamine ozidases is not yet established (Yasunobu and coworkers, 1976). Like the monoamine oxidases, diamine oxidases (histaminases) and serum amine oxidases also catalyse the oxidative deamination of amines, but purified preparations of these enzymes do not contain flavins. Evidence for the participation of pyridoxal phosphate in their action has been criticized by Suva and Abeles (1978). These amine oxidases belong to the category of Cu 2+- dependent enzymes (Yasunobu and coworkers, 1976). However, a definite interrelationship exists between the two main groups of amine oxidases. Following treatment with certain oxidizing agents, mito­ chondrial monoamine oxidases undergo 'transformation1, i.e. acquire qualit­ atively new catalytic properties, resembling the catalytic properties of diamine oxidases (Gorkin and Tatyanenko, 1967). Moreover, preparations of diamine oxidase treated with certain reducing agents acquire qualitatively new catalytic properties, resembling the catalytic properties of mitochon­ drial monoamine oxidases (Stesina and coworkers, 1971). The biological role of the amine oxidases is clearly not restricted to the inactivation of toxic amines that enter the bloodstream from the gastro­ intestinal tract. In the course of the enzymatic deamination of amines aldehydes are formed which possess very different biological activities from those of the amines (Gorkin, 1966, 1973). Amine oxidases in general, and mitochondrial monoamine oxidases in particular, 4 Amine Oxidases: Clinical Aspects carry out important regulatory functions, not only in the catabolism of neurotransmitter amines and false neurotransmitters, but also in the bio­ synthesis of amines and their reversible conversion into the storage forms of the amines (Murphy, 1978a). There are even claims in the literature of the participation of mitochondrial monoamine oxidases in the transport across membranes (particularly across the blood-brain barrier) of certain monoamines and related compounds (Cotzias and coworkers, 1974). In the early 1970s new information began to emerge on the possible role of the amine oxidases in psychiatric illness. The report of Murphy and Wyatt (1972), of decreased platelet monoamine oxidase activity in schizophrenia, attracted much attention. This decreased activity^may be genetically deter­ mined, an index of the individual's predisposition to develop mental illness of this type (Murphy and Buchsbaum, 1978). In some experimentally-induced pathological states wjiich are characterized by the accumulation of lipid peroxides, qualitative changes have been observed in the catalytic behaviour of the monoamine oxidases; these alter­ ations may provide pointers both to the pathogenesis and treatment of this group of diseases (Gorkin, 1973, 1976). The author of this book has confined himself to an assessment of contemporary work on amine oxidases of human and animal tissues, selecting from the very numerous publications on these enzymes those facts and concepts which are relevent to an understanding of their clinical significance. Such consid­ erations have largely determined the selection from the literature listed in the References section at the end of the book. Discussion of problems consid­ ered in an earlier monograph devoted to amine oxidases (Kapeller-Adler, 1970) has been avoided as far as possible. No attempt has been made to discuss the problems of priority in discovery. This monograph has been written as a handbook for the medically-orientated investigator about to start work in the field of amine oxidase research and studying their inhibitors and activators. The investigator must first decide which of the available methods he will use for estimating enzyme activity, so the next chapter deals with available methods and compares their advantages and drawbacks. For an assessment of any diagnostic and therapeutic usefulness the amine oxidases may possess, it is essential to have comprehensive information on their physicochemical properties and any available methods of purification. Even amine oxidases of bacterial and plant origin may be useful and promising material for the clinical scientist. Data, for example, on the amine oxidase from bovine dental pulp may not at first sight seem to be of interest, nor does the failure of 1 mM sodium azide to inhibit the activity of a bacterial amine oxidase appear relevant to clinical medicine. However, the amine oxidases of connective tissue, for dental pulp is an excellent connec­ tive tissue preparation, may well shed light on the nature of pulmonary fibrosis, In general, more basic information is omitted from this monograph. Such information may be found in numerous review articles (e.g. Yasunobu and coworkers, 1976). Throughout this book attention is mainly directed to the systematic and comprehensive presentation of firmly established data; such data often Introduction 5 retain their validity long after the hypotheses that prompted the experi­ ments have been forgotten. Evaluating their significance often proves mis­ leading. Some authors have tried to compare the incomparable, e.g. data on similar enzymes from different biological sources. Thus the author has avoided compiling summary tables of this kind if there were grounds for doubting the comparability of the primary information. Wherever possible, factors affecting the reproducibility of results and the comparability of data obtained in various laboratories are indicated. This information is essential for the practical application of the data, although it reduces the readability of the text considerably. However, textbooks are not written for easy reading but for detailed study. In order to become familiar with the present state of the subject and gain an insight into its development, it is quite sufficient to read the Intro­ duction, Conclusion and introductory comments to the various chapters. But readers planning to work actively in this field are recommended to study each chapter consecutively.