Pharmac. Ther. Vol. 47, pp. 391-417, 1990 Printed in Great Britain. All rights reserved

0163-7258/90 $0.00 + 0.50 © 1990 Pergamon Press plc

Specialist Subject Editor: W. KALOW

BIOCHEMISTRY A N D GENETICS OF MONOAMINE OXIDASE WALTER WEYLER,* YUN-PUNG P. H s u t ~ a n d XANDRA O. BREAKEFIELD~§ *Molecular Biology Division, VA Medical Center, San Francisco, CA 94121, U.S.A tMolecular Neurogenetics, E.K. Shriver Center, Waltham, MA 02254, U.S.A. ~Neurology Department, Massachusetts General Hospital, Boston, MA 02114, U.S.A. §Neuroscience Program, Harvard Medical School, Boston, MA 02115, U.S.A. Abstract--This chapter reviews the two mitochondrial flavin containing isozymes of monoamine oxidase. Section 1, "Biochemistry" discusses assays, substrates and inhibitors, phylogenic and tissue distribution, interactions with lipids, nutritional studies, protein structure, kinetic and chemical mechanistic proposals, and biosynthesis. Section 2, "Inheritance" discusses possible genes involved in expression, genetic studies of platelet MAO-B and fibroblast MAO-A, and chromosomal location. Section 3, "Molecular Genetics" reviews the cloning of their cDNAs, their intra- and interspecies homology and structural inferences made from deduced amino acid sequences. Section 4, "Regulation" gives an overview of levels in development and aging, and effect of drugs. The final section 5, "Role in Human Disease" discusses physiological function and effects of altered levels in humans and animal models including complete absence due to a submicroscopic chromosomal deletion in several human patients. CONTENTS 1. Biochemistry 1.1. Background 1.2. Distribution 1.3. Interaction of MAO with lipids and detergents 1.4. Substrates 1.5. Inhibitors 1.6. Assay methods 1.7. Subunit structures, FAD content and sulfhydryl groups 1.7.1. Subunits 1.7.2. FAD and active site 1.7.3. Sulfhydryl groups 1.8. Kinetic mechanism 1.9. Chemical mechanism 1.10. Biosynthesis 2. Inheritance 2.1. Genes involved 2.2. MAO-B 2.3. MAO-A 2.4. Gene location 3. Molecular Genetics 4. Regulation 4.1. Development 4.2. Aging 4.3. Hormones and drugs 5. Role in Human Disease Acknowledgements References

1. B I O C H E M I S T R Y

tertiary amines are also oxidized at a lower rate. The generalized reaction for a catalytic cycle is shown:

1.1. BACKGROUND

RR'HC-NR"R" + MAOox

The flavin containing monoamine oxidase (EC 1.4.3.4) was first described as having tyramine oxidizing activity by Mary Hare in 1928 (Hare, 1928; see Blaschko, 1972 and Kety, 1976 for a historical recount). The enzyme oxidizes primary aromatic amines, but primary aliphatic, secondary and JPT 4713--E

391 391 392 394 394 395 395 396 396 396 396 396 397 398 399 399 399 400 400 400 403 403 405 405 406 408 408

--* R R ' C = N R " R " + M A O r e d M A O r e d + 02 ~ M A O o x + H 2 0 2 R R ' C = N R " R ' " + H 2 0 ~ RR'C--O + H N R " R " 391

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Interest in this enzyme has been high for some decades since the discovery that its inhibitors show some efficacy in the treatment of depressive disorders. Correlations of high or low platelet monoamine oxidase activity with many human conditions have been claimed (for review see Denney and Denney, 1985); but further understanding of the structure and regulation of this enzyme is necesary to interpret these findings. Although a number of symposia and reviews have dealt with these issues (Costa and Sandler, 1972; Singer et al., 1979; Wolstenholme and Knight, 1976; Youdim and Paykel, 1981; Usdin et al., 1981; Gorkin, 1983; Kamijo et al., 1982; Beckmann and Riederer 1983; Mondovi, 1985; Glover and Sandler, 1986), recent findings (for review, see Hsu et al., 1989) indicate a need to re-evaluate the biochemical studies in order to understand the genetics of MAO. The selective inhibition of MAO by clorgyline (Johnston, 1968) and deprenyl (Knoll and Magyar, 1972) demonstrated the presence of two forms of enzyme. The clorgyline-sensitive form was termed MAO-A and the deprenyl-sensitive form, MAO-B (MAO will refer to both forms here). Differences between these two forms of MAO have been verified for their primary amino acid sequences, and proposed for their lipid microenvironments and post-translational modifications. Comparisons of immunological determinants, subunit sizes, and peptide maps between MAO-A and MAO-B have strongly suggested that they are unique proteins and recent cDNA cloning experiments have confirmed this. The immunological studies of MAO have been reviewed in detail (Denney and Denney, 1985); only highlights and new results will be covered here. Polyclonal antibodies raised against bovine brain MAO specifically precipitated purified bovine MAO-B (McCauley and Racker, 1973). Antibodies against bovine MAO-B yielded immunofluorescence in rat tissues and cultured cells containing MAO-B, but not MAO-A (Pintar et al., 1983a; Levitt et al., 1982). Monoclonal antibodies specific to either MAO-B from human platelet or MAO-A from human placenta provided further evidence for the existence of immunologically unique forms of the enzyme (Denney et al., 1982; Kochersperger et al., 1985; Billett and Mayer, 1986). Physical evidence that MAO-A and MAO-B were two distinct proteins was provided by differences in apparent molecular weights and peptide maps of [3H]-pargyline-labeled MAO-A and MAO-B subunits from rat cell lines (Cawthon and Breakefield, 1979, 1983), and human platelets and human placenta (Brown et al., 1980; Cawthon et al., 1981, see Fig. 1). Differences in the subunit molecular weights of purified human MAO-A and MAO-B have also been demonstrated (Weyler and Salach, 1985). Recently conclusive evidence for the independence of MAO-A and MAO-B was provided by the cloning of these enzymes from several tissues of human, bovine and rat origin (Bach et al., 1988; Hsu et al., 1988; Powell et al., 1989; Ito et al., 1988).

1.2. DISTRIBUTION

MAO appears to be ubiquitously expressed in higher eukaryotic organisms. MAO-like activity has been reported in the parasitic worms A s c a r i d i a galli and N i p p o s t r o n g y l u s braziliensis (Mishra et al., 1983; Agarwal et al., 1985), the oyster C r a s s o s t r e a a n g u l a t a (Martin del Rio et al., 1987), the cockroach (Subhashini et al., 1983), fish, birds, amphibians, and echinoderms: carp (Kinemuchi et al., 1983b, 1985; Yoshino et al., 1984); perch (Hall et al., 1982a); rainbow trout (Edwards et al., 1986); gold fish (Hall and Uruena, 1982); ring-dove (Hall et al., 1985a,b); frog (Kobayashi et al., 1981; salamander (Hall and Uruena, 1983); starfish (Nicotra et al., 1986); and sea urchin (Nicotra, 1982, Nicotra and Naccarato, 1982). A fraction of the mitochondrial MAO in frog liver behaved like a copper amine oxidase, and their FAD-containing MAO showed significantly different Km values compared to the mammalian enzymes (Kobayashi et al., 1981). MAO in carp brain appears to contain covalent FAD, and has similar molecular weight but significantly different specificity toward substrates and inhibitors as compared to mammalian MAO (Kinemuchi et al., 1983a; Yoshino et al., 1984). MAO is also found in toad eggs and embryos (Nicotra and Senatori, 1984; Senatori and Nicotra, 1985; Nicotra and Senatori, 1988) where the levels of both MAO-A and MAO-B decrease until the neural fold stage of development and then increase (Baker, 1966; Nelson and Lovtrup-Rein, 1983). A similar drop in MAO activity is also observed in sea urchin eggs and sperm from the cleavage stage to the gastrula stage, suggesting that MAO is under active regulation during embryogenesis (Nicotra and Naccarato, 1982; Nicotra, 1982). A possible role for MAO in the regulation of biogenic amines during morphogenesis has been suggested by these and other studies (see below). MAO is found in all mammals where it has been looked for. A few tissues express only one form of MAO, thus allowing the purification of MAO-A from human placenta (Weyler and Salach, 1985) and bovine thyroid (Masini-Repiso et al., 1986; Fischer et al., 1968), and MAO-B from human platelets (Denney et al., 1982), bovine kidney and liver, and pig liver (Erwin and Hellerman, 1967; Minamiura and Yasunobu, 1978; Salach, 1979; Weyler and Salach, 1981; Oreland, 1971). Subcellular fractionation and histochemical staining have been used to determine the cellular location of MAO. Microvessels from the blood-brain barrier are rich in MAO-B activity, but there is a 25-fold difference in levels among six mammalian species tested (Kalaria and Harik, 1987). Synaptosomes from guinea pig and rat brain (Fagervall and Ross, 1986) were found to contain predominantly either MAO-A or MAO-B, but not exclusively one form of MAO in any particular preparation (Ross, 1987). MAO in a single rat neuron has been studied by microgasometry and both MAO-A and MAO-B activities could be distinguished by clorgyline titration (Sket and Pavlin, 1985). Neurons from the locus coeruleus showed 60% MAO-B and 40% MAO-A activity, indicating that the same cell can express both forms of MAO. Presumably different neurons can contain various

Biochemistry and genetics of monoamine oxidase

393

0 Z Z I--

>I O.

Ld _J m

94,000

--

n" ~D 0

I-Z J J

68,000

--

58,000

--

43,000

--

Of 0

L.U

0 0 C~

30,000--i A

B

C

D

E

F

FIG. 1. Electrophoretic separation of 3H-pargyline labeled MAO-A and MAO-B. Monoamine oxidases in trophoblast mitochondria and umbilical blood platelets from the same newborn male were labeled with aH-pargyline and resolved by SDS-polyacrylamide gel electrophoresis. Gels were stained for protein with Coomassie blue (A~C) or analyzed by autoradiography (D-F). A and D, trophoblast mitochondria; B and F, platelets; C and E, mixed sample containing an approximately 1:90 ratio of trophoblast protein to platelet protein. From Cawthon et al., 1981. Reprinted with permission of the copyright holder, Raven Press Ltd, New York. levels of these two forms depending on their location, activity and neurotransmitter phenotype. Histochemical staining techniques based on specific MAO substrates and inhibitors can give extremely good resolution of the location of MAO. In the rat peripheral nervous system, MAO has been localized to the endothelial cells of the endoneurial vessels, the Schwann ceils, and neurons in some unmyelinated axons (Matsubayashi et al., 1986). Total MAO has been demonstrated in the cat hypothalamus and neurons projecting from there to the occipital cortex (Kitahama et al., 1984; for similar studies in rat see references in this paper). Mammalian adrenal glands contain variable levels of MAO with both forms expressed in the cortex and medulla, and different forms in specific cell types (Carmichael and Pfeiffer, 1985). In the human fallopian tube, the intensity of MAO staining correlates with the phases of the menstrual cycle, with the highest intensity during the secretory phase. It was suggested that MAO may protect the fertilized ovum from circulating amines (Donnez et al., 1985). Immunohistochemical studies using specific polyclonal and monoclonal antibodies have shown that serotonergic neurons contain predominantly

MAO-B and catecholaminergic neurons contain predominantly MAO-A (Levitt et al., 1982; Westlund et al., 1985). In rat brain, MAO-B is present in the cytoplasm and processes of both protoplasmic and fibrous astrocytes, and in neurons in the brainstem, raphe and hypothalamus (Levitt et al., 1982). Double-immunofluorescence experiments showed that almost all neurons that were serotonin positive were also MAO-B positive. Similarly, primate brain cells containing serotonin also stained positively with monoclonal antibody raised against MAO-B, while cells containing catecholamines stained positively only with monoclonal antibody against MAO-A (Westlund et al., 1985). These cells included those in the substantia nigra, locus coeruleus, nucleus subcoeruleus, and the periventricular regions of the hypothalamus. An improved histochemical staining technique showed similar results in cat (Kitahama et al., 1986, 1987). These results suggest that MAO-A and MAO-B are independently regulated and that their cellular distribution may underlie different functions. The observation that MAO-A and MAO-B are clearlysegregated using the immunohistochemical and high resolution histochemical methods contrast

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with that using cellular fractionation methods, where both MAO-A and MAO-B are generally present throughout fractions. This may be due to difficulties in obtaining reproducible fractions of a specific cell or particle type. The immunological methods are clearly superior for the precise localization of MAO-A and MAO-B. Recently positron emission tomography (PET) has been used to demonstrate distribution of MAO-A and MAO-B in the living human brain (Fowler et al., 1987). Now that cDNA clones for MAO are available (see below), R N A / D N A in situ hybridization techniques can also be used. The physiological roles of MAO are not entirely clear. In the gut and circulatory system it probably serves to metabolize amines that could act as false neurotransmitters. Many cell types have adrenergic receptors and systemic sources of MAO probably further serve there to regulate the hormonal actions of biogenic amines released by neurons and chromarlin cells. In neurons and neuroendocrine cells MAO probably has a more specific function in intracellular regulation of amine stores. 1.3. INTERACTIONOF MAO WITH LIPIDS AND DETERGENTS MAO is tightly associated with the outer mitochondrial membrane (Schnaitman et al., 1967). Procedures which yield pure MAO have usually involved an extraction step using a detergent or an organic solvent (Oreland, 1971). The purified enzyme must be maintained in the presence of a detergent to prevent aggregation and precipitation. However, some of the detergents used sometimes appear to cause enzyme inactivation (Achee and Gabay, 1981; Yu, 1981b; Kinemuchi et al., 1985 and Pohl and Schmidt, 1983, for other references). The batch of detergent and the handling of it can be important. For example, heating Triton X-100 in vaccuo can be used to remove volatile contaminants that may be responsible for some of the inactivation effects (Weyler and Salach, 1985). The dialyzable detergent 1-O-octyl-beta-d-glucopyranoside (OG) has also been found to inhibit MAO reversibly in some studies (Pearce and Roth, 1983; Pohl and Schmidt, 1983), but not in others (Stadt et al., 1982; Weyler and Salach, 1985; Gurne and Zeller, 1982). Detailed studies have shown that detergents can become inhibitory above a threshold concentration (Bancells et al., 1987; Kinemuchi et al., 1983b). It would therefore seem wise to check each new lot of detergent for possible inhibitory effects before use. Some phospholipids seem to be able to modify the activities of MAO. Earlier studies have assumed that only a single protein species of MAO existed and that the type of activity (A or B) was determined by the lipid environment (Houslay and Tipton, 1973b). More recently these observations have been generally interpreted in terms of separate protein species for MAO-A and MAO-B which show some sensitivity to their lipid environment (Huang, 1980). Effects of phosphatidyiserine (PS), phosphatidylinositol (PI), and cardiolipin (CL) on MAO activity in whole mitochondria or outer mitochondrial membranes have been studied, and modest changes in MAO-A activity and a 50% reduction in MAO-B activity

by PS have been observed (Buckman et al., 1983b). It is not known if this effect was due to inactivation or a change in the turnover number of the enzyme. Purified MAO exhibited altered kinetic properties with benzylamine if the enzyme was 'reconstituted' with PS or phosphatidylethanolamine (PE) (Pohl and Schmidt, 1983). In this context it is interesting that purified MAO-B from bovine liver is more stable (weeks vs days) in buffer with 0.2% Triton X-100 than in buffer without the detergent (Weyler and Salach, unpublished observation). This suggests that effects seen with the 'reconstituted' enzyme may reflect nonspecific effects of a micellar environment. Liposomes of PS, PI, or CI (Huang and Faulkner, 1981) restored rat brain MAO activity to normal or higher levels in mitochondria pretreated with phospholipase A:, which reduced 70% of the endogenous lipids and 50% of the enzyme activity, while phosphatidylcholine (PC) doubled MAO-A activity and had no effect on MAO-B activity. PE had no effect on either activity. These results contrast with those of Buckman et al. (1983a,b) who saw an inactivation effect by PS and very little effect by other phospholipids. Still different results have been obtained with other sources of M A O using other methodologies (Suh et al., 1986; Navarro-Welch and McCauley, 1982; Inagaki et al., 1986). These in vitro data are difficult to fit into a unified model. However, it is clear that MAO activity in vitro can be modulated, sometimes dramatically, by manipulating its lipid environment. A number of dietary studies are germane to this discussion. Rats on a fat-free diet have a 50% and 70% reduction in liver MAO-B and MAO-A activities, respectively, and subsequent supplementation of fats restored the activities to normal levels (Kandaswami and D'Iorio, 1979a). Rats on low-fat diets showed organ-specific changes in MAO activity with little change in the lung and the brain, but 21% and 31% reduction in the liver and the heart, respectively (Mahfouz et al., 1982). Rats on diets high in lard and low in essential fatty acids showed a 30% decrease in brain MAO-A levels (Crane and Greenwood, 1987). Altered protein-membrane interactions may underlie these changes. It is interesting to note in this context that the kinetic properties of MAO in goldfish raised at 22°C were significantly different from those raised at 7-C (Hall et al., 1982b). It seems unlikely that the lipid environment of MAO could critically regulate its activity in tissues containing a large quantity of the enzyme. However, in cells such as some neurons where the local concentration of MAO may be rate-limiting for the metabolism of a particular substrate (Waldmeier, 1987), changes in the membrane fluidity due to pathologic or normal factors may have significant effects on the activity of MAO (Orologas et al., 1986; Tachiki et al., 1986). 1.4. SUBSTRATES

The principle biogenic amines metabolized by MAO-A and MAO-B are 5-hydroxytryptamine (serotonin), N-methyl-2-hydroxy-2-(3,4-dihydroxyphenyl)ethylamine (adrenaline, epinephrine), 2-(3,4-dihydroxyphenyl)ethylamine (dopamine), 2-hydroxy-

Biochemistry and genetics of monoamine oxidase

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2-(3,4-dihydroxyphenyl)ethylamine (noradrenaline, both enantiomers of noradrenaline which has a chiral norepinephrine), 2-phenylethylamine, and tyramine center at the beta position (Williams, 1982). Stereo(see review by Waldmeier, 1987). Many amines in chemical properties of a number of MAO inhibitors addition to biogenic amines, including aliphatic, (Benedetti and Dostert, 1985) and general aspects of aromatic, primary, secondary, and tertiary amines, MAO stereospecificity have been reviewed (Williams, also serve as substrates for MAO (McEwen et al., 1982). 1968, 1969a,b). It was contended that each of the MAO isozymes 1.5. INHIBITORS acted on specific, as well as common substrates. It is A great number of MAO inhibitors has been now apparent that the specificity is relative and depends on the concentration, the affinity and reported, primarily for their potential pharmacoturnover rate of the substrate (Tipton et al., 1987). logical use. Dowson (1987) reviewed MAO inhibitors For example, 2-phenylethylamine, a substrate for in mental diseases, and Benedetti and Dostert (1987) MAO-B, can also be a substrate for MAO-A at provided a more general overview. Another review higher concentrations (Suzuki et al., 1981). Similar stressed stereochemical aspects of MAO inhibitors results for tyramine have also been reported (Fowler (Benedetti and Dostert, 1985). A broad spectrum of and Tipton, 1981). It is therefore important to use a MAO inhibitors (Wolstenholme and Knight, 1976; range of substrate concentrations to determine the Km Youdim and Paykel, 1981) and their adverse effects and Vma~, and to establish the concentration at the have also been reviewed (Blackwell, 1981). MAO catalytic center by titration with a radiolabeled inhibitors are primarily used in the treatment of acetylenic inhibitor (Tipton et al., 1987; Gomez et al., depressive illness and Parkinson disease. In the latter 1986) or by some standardized assay, so that the Kcat case, it has been found that deprenyl, given as an can be calculated and the results from different adjunct to L-Dopa, prolongs the efficacy of this drug, experiments can be compared. Since the free amines, probably due to a reduced metabolism of dopamine rather then the protonated species, serve as the generated from L-Dopa (Youdim and Finberg, 1986; substrates, it is important to adjust the Km (or Ki and Knoll, 1986). The hypertensive 'cheese effect' caused Ks) value to a standard pH for a valid comparison by the combination of using MAO-inhibitors drugs among different studies. and ingesting amine-rich foods remains a major MAO-B is involved in the conversion of the xeno- obstacle in the use of these drugs (Youdim and biotic 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine Finberg, 1987). (MPTP) to the nigrostriatum-specific neurotoxin MAO inhibitors can be divided into two categories: N-methyl-4-phenylpyridine (MPP+; see review by the reversible, keat and Vmax inhibitors with short Trevor et al., 1987). This discovery is of major duration, and the irreversible, time-dependent, importance since MPP ÷ poisoning causes brain covalent inhibitors (Richards and Burger, 1986). For lesions and clinical symptoms also associated with the recovery of MAO activity following irreversible Parkinson disease. It is hoped that animal models inhibitions de novo protein synthesis is required. developed with the use of MPTP will lead to a better Pro-MAO-inhibitors that are activated by aromatic knowledge and eventual prevention and cure of this amino acid decarboxylase to selectively reduce disease. The structure of MPTP departs from the MAO-A or MAO-B activity in the brain have also endogenous substrates for MAO in that the carbon- been developed (Palfreyman et al., 1985; McDonald nitrogen bond oxidized is part of a six membered ring et al., 1984). A new class of mechanism-based system which forms a relatively stable imine after inhibitors are the trimethylsilane derivatives of alioxidation. Many compounds which are structurally phatic (methyl through propyl) amines (Silverman related to MPTP and are possible substrates for and Banik, 1987). These compounds have relatively MAO have been investigated to identify possible high Km values for bovine MAO-B, but modifications causative agents of Parkinson disease (Singer et al., to increase their affinity seem possible and could make them of pharmacologic interest. Some of these 1988; Gibb et al., 1987). The stereochemical preference of MAO for its mechanism-based inhibitors appear to be reversible substrates has received only limited attention. with a t~/2(half-time for recovery of enzymatic activBelleau et al. (1960) showed a selective isotope effect ity) of 5.5 days for the methyl and ethyl analogs and with stereospecifically, monodeuterated tyramine. a t~/2of 13 hr for the propyl analogs. Since the moiety The pro-R hydrogen of n-heptylamine was sub- that modified the enzyme is invariant in all three sequently found to be removed during oxidation cases, differences in the t,/2 suggest that at least two (Battersby et al., 1979). Similar results have been functional groups of the enzyme interact with these inhibitors. Germanium analogs of these inhibitors reported recently for the oxidation of stereospecifically deuterated dopamine by MAO-A and MAO-B from have also been described (Silverman and Vadnere, rat liver, and MAO-A and MAO-B from placenta 1987). and platelets, respectively (Yu et al., 1986). Under anaerobic conditions S(+)-amphetamine slowly 1.6. ASSAYMETHODS reduced bovine liver MAO-B, whereas its enantiomer Assay methods for the enzyme in either purified did not (Salach and Weyler, unpublished). With Rand S-2-amino-3-fluoro-l-phenylpropane, however, or crude form were reviewed recently (Tipton, 1980; Singer, 1985). There are numerous coupled assays this stereoselectivity is not observed (Weyler et al., 1984); both of these compounds slowly reduce the based on spectrophotometric, fluorimetric and radioFAD cofactor of bovine liver MAO-B under anaerobic isotopic changes. Recent methods for special appliconditions. It is interesting to note that MAO oxidizes cations include a platelet assay for whole blood

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samples (van Kempen et al., 1985), a fluorimetric assay for brain samples (Morinan and Garratt, 1985), a rapid bioluminescence assay (Tenne et al., 1985), a monoclonal antibody radioimmunoassay (Fritz et al., 1986) and a HPLC assay for the metabolism of MPTP (Shinka et al., 1987). Catalytic centers of MAO-A and MAO-B can be estimated using radiolabeled clogyline or deprenyl (Singer, 1985; Tipton et al., 1987; Gomez et al., 1986). 1.7. SUBUNITSTRUCTURES,F A D CONTENTAND SULFHYDRYLGROUPS 1.7.1. Subunits

Based on titration using radiolabeled pargyline, it was estimated that bovine MAO-B contained one F A D per 100 kDa of protein (Chuang et al., 1974). A similar ratio was found by a spectrophotometric determination of flavin (Minamiura and Yasunobu, 1978). Based on molecular weights (55-60 kDa) from SDS-PAGE analysis, it was proposed that bovine MAO-B consists of two subunits with only one binding F A D (Minamiura and Yasunobu, 1978; see also review by Yasunobu and Tan, 1985). However, recent studies using improved MAO purification methods, quantitative amino acid analysis and dithionite flavin titration showed a ratio of one F A D per 63 kDa and 57 kDa for the human MAO-A and bovine MAO-B, respectively (Weyler, 1989). Native polyacrylamide gel analysis in the presence of detergent indicated a molecular weight of 120 kDa for monkey liver MAO (Obata et al., 1987). Other workers, however, based on gel filtration experiments have suggested that the minimum catalytic unit is 59kDa (White and Stine, 1982 and 1984b). These results, in conjunction with the molecular weights (59-60 kDa) obtained from cloned MAO (Hsu et al., 1988; Powell et al., 1989; Bach et al., 1988; Ito et al., 1988) indicate that native MAO consists of either one or more subunits, each of which binds on FAD. This is consistent with recent results that catalytically active enzyme can be expressed from a single subunit cDNA for human MAO-A or MAO-B in a COS cell line (Lan et al., 1989). 1.7.2. F A D and Active Site The covalent FAD-binding site of bovine liver MAO-B was identified by isolation of a pentapeptide, SGGCY, containing F A D linked through the cysteine residue (Kearney et al., 1971; Walker et al., 1971). The same pentapeptide was subsequently identified in other species (Nagy and Salach, 1981; Yu, 1981a) and recently identified by the cloning of human MAO-A and MAO-B (Bach et al., 1988; Hsu et al., 1988), bovine MAO-A (Powell et al., 1989), and rat MAO-B (Ito et al., 1988). For further discussion of F A D binding see also Section 3, "Molecular Genetics". The use of affinity reagents for MAO (Chen et al., 1984, 1985, 1987; Hsu and Shih, 1988; Weyler, 1987; Silverman and Zieske, 1986a) have not as yet resulted in the identification of active site residues. Chemical modification experiments give evidence for an essential histidine residue (Hiramatsu et al., 1975) and an essential carboxyl group (Weyler, unpublished).

Bovine MAO-B was not inactivated by phenylglyoxal or butanedione, suggesting that arginine residues are not essential for activity (Weyler, unpublished). 1.7.3. S u l f k y d r y l Groups The number of cysteine residues in bovine MAO-B was estimated to be eight per 100kDa of protein (Yasunobu and Tan, 1985; Erwin and Hellerman, 1967). The number of titratable sulfhydryl groups was 7 and 7.5 per mol of F A D for bovine MAO-B and human MAO-A, respectively (Weyler and Salach, 1985). Since F A D is attached to a nontitratable cysteine residue, the number should be raised to 8 and 8.5 per FAD, or 60,000-65,000g of protein (Weyler, submitted). These latter numbers are in good agreement with 9 cysteine residues per MAO-A and MAO-B subunit deduced from recent cDNA sequencing (Hsu et al., 1988; Bach et al., 1988; Powell et al., 1989). MAO from various sources is readily inactivated by sulfhydryl reagents (Erwin and Hellerman, 1967; Yasunobu and Tan, 1985) and the mechanism based inhibitor 1-phenylcyclopropylamine was shown to modify a presumed active site cysteine of bovine MAO-B (Silverman and Zieske, 1986a) Human placental MAO-A is inhibited by dipyridyldisulfide in a biphasic manner and the rate of both phases can be retarded by the competitive inhibitor d - ( + ) amphetamine (Weyler and Salach, 1985). The biphasic nature of the inhibition indicates that at least two SH groups are modified and that modification at the higher rate constant leads to only partial inactivation, thus ruling out a direct role in the catalytic mechanism for the group being modified. 1.8. KINETIC MECHANISM The steady state and presteady state kinetics of bovine MAO-B showed that there can be exceptions to the previously assumed Ping-Pong mechanism depending on the substrate oxidized (Husain et al., 1982). Ping-Pong kinetics were observed when phenylethylamine was the substrate, but benzylamine turnover involved a ternary complex with a product bound during the reoxidation of the FAD. By use of steady state kinetic methods using d-amphetamine and alternate substrates, Pearce and Roth (1985) confirmed and extended these findings. 2-Phenylethylamine, tryptamine, and tyramine were found to obey the Ping-Pong mechanism while benzylamine was oxidized in a ternary mechanism by human brain MAO-B. Recent stopped-flow experiments by Ramsay et al. (1987) suggest that with other substrates the mechanistic scheme can include product release after electron transfer to the FAD, and binding of substrate to reduced FAD. The possibility of substrate binding to the reduced form of the enzyme was suggested earlier by Pearce and Roth (1985) to explain their observation of substrate inhibition by 2-phenylethylamine at high substrate concentrations in steady state experiments. The authors suggest that 2-phenylethylamine forms a dead end complex with the free reduced enzyme. The above observations simplify proposed schemes of earlier work where noncompetitive or mixed type

Biochemistry and genetics of monoamine oxidase of inhibition by alternate substrates was thought to indicate that there were multiple substrate binding sites on human placental MAO-A (Oguchi et al., 1981; Fowler et al., 1979). Largely based on studies of the oxidation of benzylamine in the presence of various products of the reaction by membranebound, as well as solubilized and purified, rat liver MAO, Houslay and Tipton (1975, 1973b) arrived at a different kinetic mechanism. The salient features of their mechanism were that the product aldehyde (after hydrolysis of the imine on the enzyme surface) is released prior to reoxidation of the F A D and that ammonia is released after the reoxidation step. This mechanism is in contradiction to the kinetic schemes proposed by Husain et aL (1982) and Pearce and Roth (1985), and is inconsistent with the chemical and spectral evidence for imine release (Patek et al., 1972; Walker and Edmondson, 1987) prior to its hydrolysis. In an attempt to measure the effect of benzaldehyde and ammonia on the oxidative halfreaction, Husain et al. (1982) reduced the enzyme in oxygenated buffer containing a 10-fold excess of benzaldehyde and 0.1 M ammonium ion. It was found that reoxidation of the F A D was biphasic with 25% of the oxidation showing a rate constant characteristic of the free reduced enzyme and 75% with a 4-fold slower rate constant. To rationalize this result the authors suggested that aldehyde might be binding to a site other than the active site of the enzyme. Other workers have found that benzaldehyde and ammonia inhibit the steady state oxidation of benzylamine (Houslay and Tipton, 1975, 1973a; Oi et al., 1970). Based on the kinetic evidence presented, it is clear that some substrates of monoamine oxidase are oxidized in a Ping-Pong mechanism while others are oxidized in a ternary mechanism. The ternary intermediate can be either a substrate or product complex and can either accelerate or retard the oxidative half-reaction. It is also clear that additional kinetic analysis, steady state and presteady state will be useful in identifying the exact nature of product release from the enzyme and the various ternary complexes. 1.9. CHEMICALMECHANISM Despite the intense interest in this enzyme for several decades relatively little is known about its chemical mechanism. Formally the reaction is an oxidative deamination and electron transfer to the F A D is thought to proceed by a radical mechanism, hydride transfer, or oxidation of a carbanion intermediate. The reaction catalyzed by MAO consists of a reductive and oxidative half-reaction. In the reductive half-reaction the amine substrate is oxidized and the F A D is reduced to the hydroquinone. In the second half-reaction the F A D is reoxidized by molecular oxygen with the formation of H202. The primary substrate oxidation product, in the case of aliphatic substrates, is an imine which is hydrolyzed by water, presumably spontaneously, to the final product. The imine intermediate has been trapped by reduction with tritiated sodium borohydride and bovine serum albumin (BSA) for the substrates benzylamine and methylbenzylamine (Patek et al., 1972) suggesting

397

that the imine is released from the enzyme surface prior to hydrolysis to the corresponding aldehyde. In the oxidation of p-dimethylaminobenzylamine spectral evidence indicates that the imine is hydrolyzed to the final product aldehyde at a much slower rate than catalytic turnover, therefore requiring that imine be released from the enzyme (Walker and Edmondson, 1987). Primary deuterium isotope affects have been observed in steady state kinetic experiments with tyramine, kynuramine and tryptamine. The magnitude of these effects was a function of substrate and concentration (maximum effect 2.3; Belleau and Moran, 1963). Much larger isotope effects (6-7) were observed in stopped-flow and steady state kinetic investigations with benzylamine using bovine liver MAO-B (Husain et al., 1982). In these studies phenylethylamine gave an isotope effect of 3.0 in the stopped-flow experiments, but no effect in the steadystate experiments. These observations are consistent with hydrogen-carbon bond cleavage being partially rate-limiting for all of the substrates tested except phenylethylamine in the overall reaction. With the latter substrate it appears that steps after electron transfer to the flavin are rate-limiting, presumably the reoxidation of FADH2 (Husain et al., 1982). Walker and Edmondson (1987) demonstrated similar isotope effects with a number of meta- and para-substituted benzylamines in analogous studies. The details of most of the chemical steps of these mechanisms are not known. Experiments to demonstrate the involvement of a carbon ion mechanism produced negative results (Weyler, 1987). The oxidation of 2-chloro-2-phenylethylamine gave the normal oxidation product 2-chloro-2-phenylacetaldehyde and none of the beta-elimination product 2-phenylacetaldehyde. The reductive half-reaction with para- and meta-substituted benzylamines showed no linear free energy correlations and thus did not shed light on the nature of the intermediate involved in hydrogen loss (Walker and Edmondson, 1987). Silverman et al. (1980) proposed that amines are oxidized by a radical mechanism. In analogy to the electrochemical oxidation of amines it was suggested that a lone pair electron is transferred to the F A D rendering the alpha protons acidic; an alpha proton is then abstracted forming a radical anion; and a second electron is transferred to FAD. Evidence of this mechanism was presented in the studies of l-phenylcyclopropylamine (Silverman and Zieske, 1985) and 1-phenylcyclobutylamine (Silverman and Zieske, 1986b) with bovine liver MAO-B. Both compounds inhibited the enzyme, presumably by a reactive ring-opened radical intermediate. Product analysis of the reaction with 1-phenylcyclobutylamine identified 2-phenyl-l-pyrroline as a transient intermediate, which is most readily accounted for by a ring expansion mechanism via an aminium radical. The observed inhibition of MAO by (aminoalkyl)trimethylsilanes was also proposed to proceed through a radical mechanism (Silverman and Banik, 1987). Finally the similarity of products observed in the photochemical reaction of allenic and acetylenic amines with 3-methyllumiflavin (Simpson et al., 1982) and of the acetylenic amines with MAO (Macock et al., 1976) also suggests a radical as an intermediate.

398

W. WEYLERet al.

Simpson et al. (1982) proposed a kinetic mechanism which includes provision for a radical intermediate and the observed deuterium isotope effects. This model proposes that the aminium radical/flavin semiquinone pair is in equilibrium with the ES complex [ k ( 2 ) / k ( - 2 ) ] as shown in Fig. 2. In order to accommodate the apparent absence of the flavin semiquinone in the previously noted stopped-flow work (Husain et al., 1982) and more recent work (Walker and Edmondson, 1987), this model should also include the condition k(2),~ k ( - 2 ) . Large isotope effects would then be readily accounted for with k(2) ,~ k(3) and small isotope effects with k(2) ~>k(3). Proton abstraction [k(3)] may be reversible [ k ( - 3 ) ] . Transfer of the second electron to the F A D semiquinone is probably fast and irreversible. To date it has only been possible to demonstrate that both bovine MAO-B and human MAO-A can be reduced to a flavin semiquinone with the artificial reduction sodium dithionite (Weyler and Salach, 1981, 1985). Direct evidence for a radical on the catalytic reaction coordinate of MAO remains to be demonstrated. 1.10. BIOSYNTHESIS MAO from rat liver has been reported to be synthesized in ~,itro on cytosolic, free-ribosomes, rather than on membrane-bound polysomes (Sagara and Ito, 1982). This suggests that, as other mitochondrial proteins (Tzagoloff and Myers, 1986), MAO is synthesized on free ribosomes in vivo. The immunoprecipitated translation product in vitro had the apparent Mr of mature MAO, suggesting that the enzyme precursor is not processed by proteolysis. The human enzymes (Bach et al., 1988; Chen and Weyler, 1988) and the bovine liver enzyme (Minamiura and Yasunobu, 1978) appear to have a modified amino acid at the N-terminus. Transport of MAO in mitochondria within neurons has been inferred in striatal neurons from the appearance of radiolabel in rat substantia nigra seven days after injection of [3H]-pargyline into the striatum. This type of labeling combined with the use of selective inhibitors can be useful in localizing the type of MAO in a particular neuronal cell type and as a tool to study axonal transport (Gramsbergen et al., 1986). Possibly the location of MAO in the mitochondria provides a means of transport to synaptic terminals. The nature of the steps involved in the insertion of MAO into the outer mitochondrial membrane is not known. The cDNA clones for human MAO-A and MAO-B (Hsu et al., 1988; Bach et al., 1988) do not contain N-terminal sequences similar to the known

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signal sequences for other mitochondrial proteins (Sehatz, 1987). Both isozymes of MAO are tightly associated with the outer membrane of the mitochondria and have often been used as a marker enzyme for this cellular compartment (Schnaitman et al., 1967). Attempts to determine the sidedness of these enzymes are fragmentary and inconclusive. Immunological studies have suggested a vectorial orientation of MAO in the outer mitochondrial membrane (Russell et al., 1979). However, in this study the 'intact' mitochondria were lysed by > 5 5 % and the antibody was apparently not specific to MAO-A or MAO-B. Studies on the protease sensitivity of MAO bound to intact or hypotonically lysed mitochondria suggested to Buckman et aL (1984) that MAO-A and MAO-B reside on the same face of the outer mitochondrial membrane. Both forms of MAO are relatively resistant to protease degradation (Buckman et al., 1984). At a trypsin to mitochondrial protein ratio of 1:20, the half-life of the enzyme activity was about 20 rain for human placental MAO-A. At a trypsin to protein ratio of 1:5, the half-life was about 30 min for rat liver MAO-A. Beta-chymotrypsin and Staphylococcus aureus V-8 protease were less effective in reducing the enzyme activity, with half-lives in the range of hours at a 1:1 protease to protein ratio. The stability of bovine MAO-B to trypsin digestion had also been noted in other studies (Weiss and McCauley, 1979). However, the resistance of MAO to trypsin is not uniform in all species and studies (White and Stine, 1984a). As little as 10 U (1/~g) of bovine pancreatic trypsin per 1.65 mg of mitochondrial protein appreciably inactivated brain MAO-A in human, dog, rabbit and rat mitochondria. MAO-B was not affected, except in rabbit, at these trypsin concentrations. Notably, solubilized or membrane-bound enzymes from human showed equivalent sensitivity, indicating that the membrane may not contribute to the stability of MAO-B (White and Stine, 1984b). These studies clearly indicate that MAO-B is less sensitive than MAO-A to proteolysis, but they shed little light on how MAO is situated in the outer mitochondrial membrane. It is not clear whether MAO is glycosylated. Neuraminidase selectively inactivated rat liver MAO-A in hypotonically lysed mitochondria (Houslay and Marchmont, 1980). The report that pig liver MAO-B contained carbohydrates (Oreland, 1971) is in doubt because the enzyme preparation used in the study was probably not homogeneous judging from the appearance of its visible spectrum. Although cDNA sequences for MAO-A and MAO-B revealed potential N-glycosylation sites (Hsu et al., 1988; Bach et al., 1988; Powell et al., 1989), it seems

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Biochemistry and genetics of monoamine oxidase unlikely that MAO is glycosylated as the enzyme is probably synthesized on free-ribosomes (Sagara and Ito, 1982) and therefore would not be processed through the Golgi complex. The half-life for the recovery of rat liver MAO activity after irreversible inhibition by clorgyline was 3.5 and 2 days for mitochondria and microsomes, respectively (Egashira and Yamanaka, 1981). Rat skeletal MAO activity recovered with a half-life of about 6.5 days after inhibition with pargyline (Kwatra and Sourkes, 1980a). The half-lives in the brain and heart are generally longer with reported times up to 30 days (Egashira and Yamanaka, 1981; Corte and Tipton, 1980; Arnett et al., 1987). In cultured hepatocytes the half-life for the degradation of MAO is about 3-4 days, the same as that for de novo protein synthesis (Evans and Mayer, 1984). The effect of riboflavin deficiency on the activity of MAO has been studied in rats and cultured cells. In rats, deprived of riboflavin, MAO activity in the liver decreased over a period of 8 weeks from 15 to 85% (Kim and Lambooy, 1978; Kwatra and Sourkes, 1979, 1980b; Kandaswami and D'Itorio, 1979b). The brain did not lose as much activity (Dix and Lambooy, 1981), perhaps reflecting a slower turnover of the riboflavin pool or a longer half-life of the enzyme. The recovery of MAO activity following the administration of riboflavin depended on the rate of de novo protein synthesis (Kandaswami and D'Iorio, 1979b). Rats on a diet containing riboflavin analogs (the 7 or 8 methyl groups substituted by ethyl) also showed a decrease in MAO activity. After 8 weeks, animals on the 7-ethyl analog retained about 55% of the activity, while those on the 8-ethyl analog retained only 8% (Dix and Lambooy, 1981). Riboflavin, flavin mononucleotide (FMN), and F A D were not detectable in tissues following this diet, suggesting that the remaining MAO activity was being catalyzed by the analogs. Whether these analogs were covalently bound to the MAO apoenzyme is not clear (Kim and Lambooy, 1985). Currently no solid data is available on the timing of F A D addition to the apoenzyme. Preliminary in vivo experiments in rats (Smith and McCauley, 1981) suggest that incorporation of F A D into liver MAO is a relatively slow process. Incorporation of [J4C]-FAD continued for 2 hr after the animals had been treated with cycloheximide to block protein synthesis, suggesting that the rate of flavin attachment may be slower than the rate of apo-MAO synthesis. However, in flavin deprived cultures of neuroblastoma cells, return of MAO activity following readdition of flavin is dependent on de novo protein and R N A synthesis (Bonefil et al., 1981). It is not clear at this time whether covalent attachment of F A D to MAO is a cotranslational or post-translational process. Iron deficiency in rats also leads to reduction in MAO activity (Sourkes, 1979; Kwatra and Sourkes, 1979). Highly purified bovine liver enzyme, however, does not contain a significant amount of iron (Weyler and Salach, 1981; Ichinose et al., 1982). The role of iron in regulating MAO activity remains speculative (see Sourkes, 1979 for references). There is only a single report which may be a clue to the biodegradative fate of F A D bound to MAO.

399

The compound 8-alpha-sulfonylriboflavin has been identified in human urine and milk, and bovine milk and may arise from endogenous turnover of MAO (McCormick et al., 1987).

2. I N H E R I T A N C E 2.1. GENESINVOLVED Several types of genes might be involved in the regulation of MAO activity in vioo. These include structural genes for the two forms of MAO, genes encoding regulatory factors which might modulate levels of transcription of the M A O loci: genes involved in post-translational modification of the enzymes, such as flavin-addition, glycosylation and insertion into the outer mitochondrial membrane; and genes controlling the microenvironment of MAO, such as those involved in lipid metabolism. Since most of these genes have not been identified, any discussion of the inheritance of MAO must be presented in phenomenologic terms. Some variations in levels of MAO activity may result from allelic differences in structural genes for these enzymes. In fact, human life is tolerant even to complete loss of MAO genes and activity (see Section 4). Evidence to date indicates that at least one subunit each of MAO-A and MAO-B is encoded in separate genes (Powell et al., 1989; Bach et al., 1988). It has yet to be established whether each form of the enzyme consists of one or two subunits; and if two, whether they are identical and encoded in the same gene. If an individual's level of MAO-A and MAO-B activities are determined primarily by the structural genes for these enzymes then there should be a correlation between levels observed in peripheral cells, such as fibroblasts and platelets, and those in the nervous system. Such a correlation would justify peripheral measurements as a 'window' of brain amine metabolism. Although studies to date have demonstrated only two forms of MAO, A and B, throughout the human body, they do not exclude the possibility of other rare forms and hence other structural genes may exist. Most studies of the inheritance of MAO have been carried out in humans, but several studies indicate that up to 50% variations in total activity can occur among inbred strains of mice (MacPike and Meier, 1976; Kellog, 1971). 2.2. MAO-B Most analyses of human MAO have been carried out using platelets which express only the B form of the enzyme. Large studies in control, adult populations indicate that for a given individual the level of activity is fairly constant over time (Murphy et al., 1976b). There is a unimodal distribution of activity for males and females, with a range of over 50-fold between extremes. Females have ~ 20% higher levels than males, and in both sexes there is a small increase with age over 60 years. Levels of platelet MAO-B activity are to a large extent genetically determined. Interclass correlation

400

W. WEYLERet al.

coefficients are 0.76-0.88 for monozygotic twins and 0.39-0.45 for dizygotic twins (Wyatt et al., 1975; Nies et al., 1973); heritability has been calculated at about 0.8 (Nies et al., 1973); and variance among families is much higher than within families, 1.06 and 0.36, respectively (Gershon et al., 1980). Segregation analysis indicates that a single major locus underlies about 30% of the variation among individuals with a polygenic background accounting for the rest. The predominant major allele in humans appears to underlie the 'low activity' phenotype (Rice et al., 1984). It is difficult to distinguish between autosomal and X-linked inheritance by these statistical models (Goldin et al., 1982). Since structural genes for MAO-A and MAO-B appear to be on the X-chromosome (Pintar et al., 1981b; Kochsperger et al., 1986; Sims et al., 1989a), some component of heritable activity should be X-linked. There is no direct evidence to date for structural variations in MAO-B among humans. Some differences in activity can be accounted for by differences in the levels of active enzyme molecules, as determined by titration of active sites with labeled pargyline (Giller et al., 1982). Differences in K m values among low and high variants of MAO-B activity are relatively small, and no differences in the isoelectric point of [3H]-pargyline-labeled enzymes has been observed (Giller et al., 1982). Others have reported variations in the catalytic activity of enzyme molecules among individuals, as determined by combined kinetic and immunotitration studies (Rose et al., 1986). Thermolabile forms of MAO-B have also been described in control individuals, and this variant property appears to be heritable (Bridge et al., 1981). Alcohol-sensitive forms of MAO-B have been reported and appear to have a higher frequency in alcoholics; it is not clear, however, whether this is a genetically determined trait or a secondary effect of alcohol consumption on the enzyme (Tabakoff et al., 1988). All these findings suggesting possible variations in MAO-B structure are indirect and await analysis at the gene level. In fact, no correlation has been found for the levels of MAO-B in platelets and brain from the same individual (Oreland, 1979; Young et al., 1986), supporting the notion that genetic factors, other than the structural gene itself serve to regulate levels of MAO-B in humans. 2.3. MAO-A Cultured skin fibroblasts are the only readily available tissue source from living humans that expresses MAO-A activity. Levels of total MAO activity in these cells are quite low: proportions of MAO-A:MAO-B activity vary from 80-100% :20-0%, respectively, among lines (Edelstein et al., 1978; Edelstein and Breakefield, 1986). If culture conditions are controlled, constant levels of activity are observed among passages of the same cell line during the proliferative phase of its growth in culture (Breakefield et al., 1981). Values vary over 50-fold among lines from controls of comparable age, with no notable differences between males and females. Values measured in cells from monozygotic twins are highly correlated, indicating a strong genetic determinant (Breakefield et al., 1980). The advantage of measure-

ments in fibroblasts is that the environment can be held constant, thus making differences in MAO activity inherent to the genetic make-up of the cells. The disadvantage is that the intrusive nature of the skin biopsy and time-consuming requirements of cell culture have limited the number of studies done, and no values on large families are available to assess the pattern of inheritance. Again there is no evidence for structural variations in the MAO-A enzyme. Clorgyline inhibition curves indicate that levels of activity are proportional to the number of active enzyme molecules among individual lines, at least for those with relatively high levels of activity (Costa et al., 1980). Similarly there is no evidence for temperature-sensitivity or substrateaffinity variants. The very low levels of activity in some lines had made it difficult, however, to assess such properties of the enzyme. None of these analyses exclude the possibility that structural variants of MAO-A exist; analysis of allelic variations in the M A O A gene should help to resolve this. 2.4. GENE LOCATION Preliminary studies on the chromosomal location of genes for MAO-A and MAO-B were carried out using somatic cell hybrid lines containing different human chromosomes against a rodent background. Pintar et al. (1981a) analyzed hybrids prepared between normal male human fibroblasts and mouse neuroblastoma cells, which lacked MAO and hypoxanthine phosphoribosyltransferase activities. Expression of the human type of MAO-A was established by partial proteolysis and peptide mapping of molecules irreversibly labeled with [3H]-pargyline. Expression of MAO activity correlated with the presence of part or all of the human X-chromosome, as determined by karyotypic analysis and expression of HPRT activity. Further studies using monoclonal antibodies specific for human MAO-B have demonstrated that an antigenic determinant for this form of MAO, presumably the structure gene itself, is also on the human X-chromosome (Kochersperger et al., 1986). In these latter studies somatic cell hybrids between human fibroblasts and mouse neuroblastoma cells (see above), as well as hybrids between human fibroblasts and mouse hepatoma cells were used. The X-linkage of human MAO-A and MAO-B genes has been confirmed by molecular genetic analysis (see Section 3). Other studies using mouse x rat hybrid cells, again with the mouse line lacking MAO activity, demonstrated that expression of both MAO-A and MAO-B activities depends on the presence of the rat X-chromosome (Pintar et al., 1981b). Presumably the genes for MAO are on the X-chromosome in a number of mammalian species, as this chromosome tends to maintain the same set of genes across species.

3. M O L E C U L A R GENETICS Many questions regarding the structure and genetics of MAO can only be resolved by determining the primary amino acid sequences of these proteins, as well as the number and the organization of the structural genes. Recent results from cDNA clones

401

Biochemistry and genetics of monoamine oxidase

been characterized (Fig. 3). A conserved region among all these clones includes an ADP-binding site near the N-terminal regions (residues 15--45 for M A O - A ) which has features of the F A D binding sites found in a number of other flavin-containing enzymes

provide some of this information (for review see Hsu et al., 1989). Full length c D N A clones for human M A O - A (Hsu et al., 1988; Bach et al., 1988), bovine M A O - A (Powell et al., 1989), human M A O - B (Bach et al., 1988) and rat M A O - B (Ito et al., 1988) have

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351 GFILARKADR L A I ~ I R K RKICERYAKV LGSQEALHPV HYEEKNWCOE BMAO-A 351 :::::::::: :::L::E::: K::::L:::: :::::::::: ::::::::E: HMAO-A :::::::E: 194A.O-B 342 ::::H::RK ::RLT:EE:L K:L::L: :: :::L:::E:: RMAD-B BMAO-B\P : :L:DL: i] i~ii :::::::E: •





.

401 QYSGGCYTAY FPPGIMT~YG RVIP~PVGRI I'FAGTETATO WSGYMEGAVE BMAO-A 401 :::::::::: :::::::::: :::::::::: ¥::::::::K :::::::::: HMAO-A I~4AO-B 392 ::::::::T: :::::L:::: ::L::::D:: :::::::::H RMAO-B BMAO-B\P :::::::::: :: : :::::::::h *

*

**

*

*

*

BMAO-A HMAO-A 194AO-B RMAO-B BM~D-B\P m4AO-A\P

451 A G ~ NALGKLSAKD IWIOEPEAED VPAVEITPSF WERNLPSVSG ,51 !!ii!!ili! :G:::VTE:: ::V::::SK: :::::HT: 442 H:M::IPEDE ::OS:::SV: I~I:TT: ~ilhiiii~ H:I::IPEDE ::~P:::SV: :NT: L::H::::P: ~42 ....... •:I~ H:M::LPEDE ::LP:::SV: :KP:ST:S Mmm •

BMAO-A HMAO-A I~4AO-B RMAO-B

501 LLKIVGFST- -SITALWF91~ YRFRLLSRS 501 :::I::::- -:V:::G::L :KYK::P:: 492 :RLI:LT:I F:A:::G:LA HKRG::V:V ~92 ::LL:LT:I L:A:::G:LA HKKG:FV:F

::: H : V : : *

**

*

* %

FIG. 3. Comparison of amino acid sequences for human, bovine, and rat MAO-A and MAO-B. Bovine MAO-A (BMAO-A), human MAO-A (HMAO-A), human MAO-B (HMAO-B) and rat MAO-B (RMAO-B) sequences were derived from cDNA clones (PoweU et al., 1989; Hsu et al., 1988; Bach et al., 1988; Ito eta/., 1988). Bovine MAO-B peptide (BMAO-BP) and human MAO-A peptide (HMAO-AP) sequences were derived from purified enzymes (Chen and Weyler, 1988; Powell et al., 1989), with ' > ' designating the start of each peptide. Asterisks indicate the positions where amino acids are specifically conserved for MAO-A and MAO-B. From Hsu et al., 1989. Reprinted with permission of the copyright holder, Raven Press Ltd, New York.

402

W. WEYLERet al.

(Arscott et al., 1982; Wierenga et al., 1986; Weyler et al., 1987). This region is presumably involved in binding of FAD at the active site of MAO. There is a highly conserved region (residues 389-460 for MAO-A) near the C-terminal of these proteins which bears within it the covalent cysteine attachment site for FAD. A third conserved region (residues 187-230 for MAO-A) of unknown function is probably also part of the active site. Another interesting feature of these proteins is the presence ofa hydrophobic region (residues 504-521 for MAO-A) that is flanked by positively charged amino acids near the C-termini. On the basis of its similarity to the N-terminal sequence for targeting and anchoring the 70 kDa yeast protein to the outer mitochondrial membrane (Hase et al., 1984), this C-terminal end of MAO may be important in directing it to its cellular location. In fact there is little else in the sequence of the MAO proteins to indicate how they associate with membranes, since there are no other long hydrophobic stretches. This sequence information suggests that MAO may not be an 'integral' membrane protein, but rather may be transposed to the other side of the outer mitochondrial membrane with only a portion of it attached to this membrane. Separate genes encode subunits for MAO-A and MAO-B, as is apparent from discrete and scattered differences in amino acid sequences between bovine MAO-A and MAO-B (Powell et al., 1989) and between human MAO-A and MAO-B (Bach et al., 1988) with an overall homology of 71%. The evidence for distinct A and B activities in the toad (Nicotra and Senatori, 1988) indicates that duplication of the M A O gene occurred fairly early in evolution. The existence of separate genes for MAO-A and MAO-B and their differential expression indicates they are under separate regulatory controls at the transcriptional level. cDNAs for human and bovine MAO-A encode a protein of 527 amino acids, molecular mass 59.7 kDa. Two mRNA species for MAO-A have been detected of 4.4-5.4 kb and 2.1 kb; these messages differ, at least in part, by variations in sites of polyadenylation (Hsu et al., 1988) (Fig. 4). The cDNA for human MAO-B encodes a protein of 520 amino acids, molecular mass 58.8 kDa, about 1 kDa smaller than that for MAO-A (Bach et al., 1988). A single mRNA species for MAO-B of 2.8-3.1 kb has been detected in human and bovine tissues (Bach et al., 1988; Powell et al., 1989). The question has not been resolved as to whether separate genes and/or mRNAs differing in coding sequences exist for each of the subunits of MAO-A and MAO-B, nor in fact whether there are two distinct subunits for each of these enzymes. Further work on sequencing additional cDNAs for MAO-A and MAO-B should resolve whether multiple subunits exist, keeping in mind that there may be allelic polymorphism at any given gene locus. There are substantial nucleotide differences in the 5j and 3' noncoding regions of mRNAs for human MAO-A, and a few in the coding region as determined by two groups; while the encoded amino acid sequences are identical (Hsu et al., 1988; Bach et al., 1988). Differences at the 5' and 3' ends may reflect variability in the sites of initiation of transcription, splicing modes,

--5.3

Kb

- - 2 . 8 Kb

-,- 2.0 K b

1

2

3

4

FIG. 4. Bovine mRNAs for MAO-A and MAO-B. Total (lanes 1 and 2) and poly A+ (lanes 3 and 4) RNA were extracted from bovine brain (lanes l and 3) and liver (lanes 2 and 4) and resolved by electrophoresis in denaturing, agarose gels. Following Northern blotting, filters were hybridized to a 0.57kb PvulI fragment of the bovine cDNA clone for MAO-A, 34-3A, which contains sequences encoding the conserved, covalent FAD attachment site, as described (Powell et al., 1989). Levels of MAO activity in parallel samples were determined to be 268 pmol/min/mg protein (90% A and 10% B) in brain, and 2578pmol/ min/mg protein (98% B) in liver, From Powell et al., 1989. Reprinted with permission of the copyright holder, the Biochemical Society, London. and/or sites of polyadenylation, as well as the possibility of multiple genes for MAO-A or errors in sequencing. Clearly to understand the structure and inheritance of the MAOs, these issues need to be resolved. A structural gene for MAO-A has been assigned to the p arm, region 11.23-11.4, of the human X-chromosome (Fig. 5) using a full length cDNA probe for this gene and several mapping techniques: Southern blot analysis of genomic DNA from individuals containing varying numbers of human X-chromosomes and Xp deletions, and from somatic cell hybrids retaining varying portions of the human X-chromosome (Ozelius et al., 1988; Sims et al., 1989); linkage analysis in large reference pedigrees (Ozelius et al., 1988; Haines et al., in preparation); and in situ hybridization to metaphase chromosomes

Biochemistry and genetics of monoamine oxidase

403

2:1.3 ~.1

I oxm

21.3 21.2 21.1 11.4

II DXS7 NDP

11.3 I 1.23 11.~'J 1121 11.1 11 12.1 12.2

! OTC/~I.S IdAOI IDXS!

13

22.1 222 22.3 22.1 ~.2 223 23 24 26 26

27 28

X FIG. 5. Location of M A O A gene on human X-chromosome. Drawing of Giemsa-banded human X-chromosome at metaphase showing locations within which genes lie. M A O A encodes MAO-A; 0 TC encodes ornithine transcarbamylase; 55.5, DXS28 and D X S 14 refer to random, unique sequences. From Ozelius et al., 1988. Reprinted with permission of the copyright holder, Academic Press, San Diego.

(Levy et al., 1989; Lan et al., 1989). Two restriction enzymes EcoRV and MspI reveal polymorphic sites at the M A O A locus, using cDNA clone H M I I or genomic clone A2R/F2 and genomic clone A2R/D7, respectively (Fig. 6; Ozelius et al., 1988; Ozelius et al., 1989). This makes M A O A a highly informative locus with a PIC value (Botstein et al., 1980) of 0.61. Linkage analysis using reference pedigrees places this locus about 10 cM from O T C , toward the p terminal end of the X-chromosome, and 14 cM from O A T L 1, toward the centromere (Haines et al., in preparation). No recombinations between M A O A and the random D3($7 locus have been detected with a current lod score at theta (0) of +23 (Haines et al., in preparation). The nearness of these latter two loci and their combined loss in Norrie patients with a submicroscopic deletion in this region of the X-chromosome (Sims et al., 1989b) support the potential usefulness of haplotypes at the M A O A locus in genetic counselling for this X-linked disease with features of blindness, deafness and mental retardation. The concurrent loss of all genomic fragments recognized by the cDNA for MAO-A, the absence of MAO-B activity in platelets and the accumulation of MAO-B substrate phenylethylamine (Murphy et al., 1989) in these patients suggests that the M A O B gene is also

lost and therefore lies close to the M A O A locus (see Section 4). Recent in situ hybridization places the M A O B locus very close to M A O A (Lan et al., 1989).

4. R E G U L A T I O N A large number of factors appears to regulate levels of MAO-A and MAO-B including developmental changes, hormones, diet and aging. Although MAO activity is expressed in all cells tested, levels vary over 1000-fold among cells and tissues, and up to 50-fold variations within a cell type or tissue have been reported during development and in response to hormones. Very little information is available as to what molecular mechanisms might underlie this regulation. An overview of conditions affecting MAO activity will be provided here, but only a few of the many articles on this subject will be cited. 4.1. DEVELOPMENT During development in most mammalian species studied--human, rat and mouse, the A form of MAO appears before the B form in most tissues. MAO-A is almost at adult levels at birth, and undergoes a

404

W. WEYLERet al.

-&

B kb

4---10.2

k.b.b

B1---~

4---9.5

B2.--~

4--4.0

4--6.5

.4---3.7

1

2

3

1

2

3

FIG. 6. DNA polymorphisms at the human M A O A locus. Genomic DNA from lymphoblasts was digested to completion with EcoRV (panel A) and MspI (panel B). Following electrophoresis and Southern transfer, filters were hybridized to human cDNA clone HMI1 or the human genomic clone A2R/D7 (see legend to Fig. 4) Arrows indicate polymorphic fragments. Panel A, lane 1 shows an individual homozygous for the A1 allele; lane 2, heterozygous for AI and A2 alleles; lane 3, homozygous for the A2 allele. In panel B the heterozygote is shown in lane 3 with homozygotes for the B2 and BI alleles in lanes 1 and 2, respectively. From Ozelius et al., 1988, 1989. Reprinted with permission of the copyright holders, Academic Press, San Diego and IRL Press Ltd, Oxford.

1.5-2-fold increase postnatally (Tsang et al., 1986; Lewinsohn et al., 1980; Diez and Maderdrut, 1977). In the developing rat CNS the increase in M A O - A follows the caudal-rostral pattern of neuronal differentiation (Tsang et al., 1986). A marked exception to this slow increase in A activity is a 30-fold increase in rat heart from 3 to 8 weeks postnatally (Edwards et al., 1979). MAO-B activity in these species increases several-fold after birth in most tissues of the body; in contrast, it rises even more dramatically-2-5-fold in the brain during this period and is already at high adult levels in the liver at birth (Tsang et al., 1986; Lewinsohn et al., 1980; Diez and Maderdrut, 1977). The large increase in brain MAO-B is attributed to the proliferation of astrocytes in the brain shortly after birth, as most MAO-B in the brain is localized to this cell type (Levitt et al., 1982; Westlund et al., 1985). No increase is found after birth in the guinea pig brain where astrocyte proliferation occurs prior to birth (Banns et al., 1980). Changes in MAO activities during development probably reflect events of cellular differentiation, as well as the changing ratios of different cell types within tissues. Studies in cell culture support the expression of specific forms of MAO by differentiated neural cell types. Catecholaminergic neurons in vivo, at least in rats and humans, express high levels of MAO-A activity and undetectable levels of MAO-B activity (Goridis and Neff, 1972; Demarest et al., 1980). It appears that essentially all neurons with high tyrosine hydroxylase (TH) activity also have high MAO-A

activity. Consistent with this correlation, pheochromocytoma line PC12 derived from the rat adrenal medulla expresses TH and MAO-A activities (Youdim et al., 1986; Lenzen et al., 1987). However, cultured bovine chromaffin cells with TH activity express predominantly MAO-B activity (Youdim et al., 1984; Carmichael and Pfeiffer, 1985). In culture, sympathetic neurons express high levels of TH and MAO-A, and when the phenotype of these neurons is changed to cholinergic by exposure to heart-conditioned medium, levels of both these enzymes drop markedly, while levels of MAO-B remain low (Pintar et al., 1987). Similarly, explants of chick sympathetic ganglia respond to nerve growth factor with increased numbers of neurons and MAO-A activity (Phillipson and Sandler, 1975). Levels of MAO-B activity increase in cultures of hybrid cells NGI08-15 (formed by the fusion of N18 mouse neuroblastoma cells and C6 rat glioma cells) after treatment with dibutyryl cyclic AMP, an agent which promotes cholinergic differentiation of these cells (Nakano et al., 1985). Since N18 and C6 cells express only MAO-A activity (Hawkins and Breakefield, 1978; Murphy et al., 1976a) the basis for this increase is not clear. Some increase in MAO-B activity is also observed in cultures of primary astrocytes with time in culture and in response to dibutyryl cyclic A M P (Yu and Hertz, 1982). It appears then that expression of MAO-A and MAO-B responds differently to developmental signals in culture. A notable example of this is the transient expression

Biochemistry and genetics of monoamine oxidase of amine storage and MAO-B activity in the yolk sac, gut endoderm, neural tube and notochord of developing quail embryos (Levitt et al., 1985). 4.2. AGING The process of aging also appears to involve changes in levels of MAO activity. Increases in human brain MAO-B activity have been observed in several studies (Robinson et al., 1972; Shih, 1979; Fowler et al., 1980b); both increases (Shih, 1979) and no change (Fowler et al., 1980a) have been reported for MAO-A. In Shih's study (1979), MAO-B activity increased about 50% while MAO-A activity rose 2-3-fold in a linear manner from 20 to 80 years of age in males. This apparent increase in MAO activity in brain with age occurs concomitantly with an overall decrease in the activity of biosynthetic enzymes for catecholamines; together these changes probably account for decreases in norepinephrine and dopamine in the human CNS with age (Pradhan, 1980; McGeer and McGeer, 1980). Studies of MAO in rodent models of aging have been even more limited, but a recent study observed essentially no change in MAO-A or MAO-B activities in rat CNS from 2 to 28 months of age (Leung et al., 1981). Several laboratories have reported, however, a dramatic increase in MAO-A activity in rat heart with age which appears to result at least in part from a reduced rate of enzyme turnover (Corte and Callingham, 1977; Edwards et al., 1979). An increase in MAO-A with aging is supported by studies on cultured human skin fibroblasts. The specific activity of MAO-A assessed during proliferative growth of these cells in culture showed an overall increase of 5-10-fold in males from 1 to 60 years (Breakefield et al., 1980). This phenomenon was mimicked in several lines tested as cells approached the end of their proliferative life-span in culture (senescence; Breakefield et al., 1981). Only about a 2-fold increase in MAO-B activity was found in one line compared during the proliferative and senescent phase of growth (Edelstein and Breakefield, 1986). These increases in MAO-A activity may reflect the same events underlying age-related changes in mitochondria morphology (Goldstein et al., 1984; Vanneste and de Aquilar, 1981) and lipid content (Miquel et al., 1980; Nohl and Heger, 1978; Grinna, 1977). No dramatic or consistent changes with aging have been reported, however, for a number of other mitochondrial enzymes or for mitochondrial protein content in rat heart (Nohl et al., 1979) or brain (Ryder, 1980). It is possible then that the MAO protein is responding to a change in its environment during aging, but it is also possible that the M A O A gene is being regulated by transcriptional factors involved in the aging process. 4.3. HORMONESAND DRUGS A large number of studies have been carried out to assess the effects of different hormonal states and drugs on MAO activity. Interpretation of this literature is complicated by the facts that differential determinations of MAO-A and MAO-B activities have usually not been carried out and that conflicting

405

results have been reported from different laboratories for the same tissue, and from the same laboratory for different tissues (for overview see Edelstein and Breakefield, 1986; Glover and Sandier, 1986). Since many hormones can exert their actions at the gene level, however, these studies may provide clues to understanding transcriptional regulation of the MAO genes. It must be kept in mind, however, that changes in MAO activity in vivo may be secondary to other physiologic changes caused by hormonal perturbations. Variations in activity are usually in the order of 20-50%; the few representative studies reported here were done in rats. Hypophysectomy, or loss of pituitary hormones, causes both increased and decreased MAO activity in different tissues of the same animal (Illsley et al,, 1980; Youdim and Holzbauer, 1976; Clarke and Sampath, 1976). Ovariectomized females show no apparent change in MAO activity, but subsequent administration of testosterone, hydrocortisone or progesterone can increase activity in the uterus (Grosso and Gawienowski, 1975) and administration of estradiol can increase MAO-B and decrease MAO-A activities in brain (Chevillard et al., 1981; Luine et al., 1975). This latter decrease appears to be due to an increased rate of degradation of active MAO-A molecules (Luine and McEwen, 1977). Castration leads to 3-fold increases in MAO-A activity in liver with no change in MAO-B activity, and this increase can be prevented by administration of estradiol or testosterone (Illsley and Lamartinielli, 1980). It appears then that administration of estradiol directly or indirectly depresses MAO-A activity and elevates MAO-B activity. Progesterone has an elevating effect on MAO-A and MAO-B. Varying levels of these two hormones may contribute to fluctuations in MAO activity observed during the estrus and menstrual cycles (Holzbauer and Youdim 1973; Collins et al., 1970). Adrenalectomy leads to an increase in MAO activity in a number of tissues and this increase can be blocked by administration of glucocorticoids (Sampath and Clarke, 1972; Parvez and Parvez, 1973; Clarke and Sampath, 1976). In a parallel set of studies, treatment with metyrapone, which blocks glucocorticoid synthesis, also increased MAO activity (Parvez and Parvez, 1973). This apparent depression of MAO activity in response to glucocorticoids in vivo stands in sharp contrast to its effects in culture (see below). Creation of a hypothyroid state by treatment with propylthiouracil causes a dramatic increase (about 9-fold) in brain MAO activity (Battie and Verity, 1979) which appears to be mediated in part by alterations in mitochondrial biosynthesis (Jakovcic et al., 1978). Drugs interacting with catecholamines have also been reported to affect MAO activity: reserpine and L-DOPA can increase both catecholamine levels and MAO activity (Izumi et al., 1969; Lyles, 1978; Collins et al., 1970); and a number of psychoactive drugs, e.g. phenelzine, amphetamine and chlorpromazine, can depress MAO-B activity in platelets (Murphy, 1976). Studies in vivo suggest that MAO activities are finely regulated in different cells in response to varying physiologic states. A limited number of studies have been carried out in cultured cells to explore these

406

W. WEYLERet al.

phenomena further. When explant cultures of superior cervical ganglia, which contain both neuronal and non-neuronal cells, were treated with high potassium concentrations (presumably mimicking neuronal depolarization), M A O activity increased about 50% ( G o o d m a n et al., 1974). This increase is presumed to be of the A type as T H and dopamine betahydroxylase activities also increased. Pintar et al. (1987) observed a decrease in M A O - A activity in pure adrenergic neuronal cultures from this ganglia treated with heart-conditioned medium. Several lines of evidence suggest then that adrenergic neurons use primarily the A form of M A O to regulate endogenous catecholamine stores and that levels of M A O - A may respond to the physiologic activity of these neurons. More extensive studies have been carried out on hormonal regulation of M A O in cultured human skin fibroblasts. These cells offer the advantage of a pure primary cell type that can be grown in serum-free medium so that one hormone can be assessed at a time. A preliminary screen revealed that short (6 hr) exposures to several hormones raised total M A O activity in these cells; the hormones tested included progesterone, testosterone, triiodothyronine and the glucocorticoid, dexamethasone (Edelstein and Breakefield, 1981). Further studies on dexamethasone treatment, which caused the greatest increase, revealed a 6-14-fold increase in active M A O - A molecules and a 2-3-fold increase in active M A O - B molecules over a 9 day exposure to 50 nM dexamethasone (Table 1) (Edelstein and Breakefield, 1986). Studies on the synthesis, degradation and half-life of M A O indicated that dexamethasone acted by increasing the rate of synthesis of active M A O molecules. Subsequent

experiments using a c D N A for M A O - A have demonstrated a dramatic increase in the corresponding m R N A in response to dexametbasone (Sims et al., 1989b). This effect could be mediated by changes in the rates of transcription, processing or degradation of this m R N A . In contrast, no change in M A O - A activity was observed in a continuous line of rat pheochromocytoma P C I 2 cells in response to dexamethasone (Utterback and Breakefield, personal communication), although these cells do respond to glucocorticoids in other ways (Saban et al., 1983). Most enzyme inductions mediated by glucocorticoids act by transcriptional regulation through consensus sequences in the 5' region of genes which bind the glucocorticoid receptor (Martinez et al., 1987: Y a m a m o t o , 1985). It is possible that such sequences exist in front of the M A O A , but not the M A O B gene. It is difficult to reconcile these dramatic results in culture with the generally depressive effect of glucocorticoids on M A O activity observed in vivo. This discrepancy may point to a secondary means through which glucocorticoids act in vivo. Now that genomic sequences for the M A O genes are being characterized, it should be possible to elucidate transcriptional elements involved in regulation of M A O in response to differentiation, hormone action and neuronal activity.

5. R O L E IN H U M A N

DISEASE

Several lines of evidence suggest that hereditary variations in gene loci controlling levels of M A O activity may cause or contribute to human disease conditions, but no causal connections have yet been

TABLE 1. Effects of Dexamethasone and Cellular Aging on Activity, Concentration, and Molecular Turnover Number o f M A O *

Type of activity MAO A + B MAO A MAO B

Dex + + +

V~×t

[MAO]~

MTN§

Early Late (pmol/min/mg)

Early Late (fmol/mg)

Early Late (tool/tool/rain)

95.4 799.7 68.4 695.2 36.4 64.6

241 2546 148 1571 79 146

522.4 1715.0 383.2 1487.5 75.3 143.9

1340 4759 892 3415 160 323

396 314 459 443 459 441

390 360 429 436 469 446

*Crude mitochondrial fractions were isolated from nonsenescent (early; passage 6) and senescent (late; passage 11) cultures of HF27 exposed to serum-free medium with no hormone ( - ) or 50 nM dexamethasone (+) for 9 days, as described by Edelstein and Breakefield, 1986. tEstimated maximum velocities (Vm,x) were calculated by correcting MAO activities in mitochondrial fractions for subsaturating tryptamine concentrations. Results are expressed as pmol tryptamine deaminated/min/mg mitochondrial protein. Each value represents the average of two experiments in which mitochondrial fractions were assayed in triplicate at one protein concentration (values differed

Biochemistry and genetics of monoamine oxidase.

This chapter reviews the two mitochondrial flavin containing isozymes of monoamine oxidase. Section 1, "Biochemistry" discusses assays, substrates and...
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