Molecular characterization of monoamine oxidases A and B.

Monoamine oxidase A and B (MAO A and B) are the major neurotransmitter-degrading enzymes in the central nervous system and in peripheral tissues. MAO A and B cDNAs from human, rat, and bovine species have been cloned and their deduced amino acid sequences compared. Comparison of A and B forms of the enzyme shows approximately 70% sequence identity, whereas comparison of the A or B forms across species reveals a higher sequence identity of 87%. Within these sequences, several functional regions have been identified that contain crucial amino acid residues participating in flavin adenine dinucleotide (FAD) or substrate binding. These include a dinucleotide-binding site, a second FAD-binding site, a fingerprint site, the FAD covalent-binding site, an active site, and the membrane-anchoring site. The specific residues that play a role in FAD or substrate binding were identified by comparing sequences in wild-type and variants of MAO with those in soluble flavoproteins of known structures. The genes that encode MAO A and B are closely aligned on the X chromosome (Xp11.23), and have identical exon-intron organization. Immunocytochemical localization studies of MAO A and B in primate brain showed distribution in distinct neurons with diverse physiological functions. A defective MAO A gene has been reported to associate with abnormal aggressive behavior. A deleterious role played by MAO B is the activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a proneurotoxin that can cause a parkinsonian syndrome in mammals. Deprenyl, an inhibitor of MAO B, has been used for the treatment of early-stage Parkinson's disease and provides protection of neurons from age-related decay.

Arginine-42 and threonine-45 are required for FAD incorporation and catalytic activity in human monoamine oxidase B.

Monoamine oxidase B (MAO B) is an integral protein of the outer mitochondrial membrane that is involved in the deamination of vasoactive and neuroactive amines. The oxidation of these amine substrates requires the cofactor FAD, which is covalently bound to Cys-397 of human MAO B. Previously, Glu-34 and Tyr-44 of MAO B have been identified as residues which engage in noncovalent interactions with FAD that are required for subsequent covalent FAD binding and generation of catalytic activity. In this study, we have identified two additional residues, Arg-42 and Thr-45, which form noncovalent contacts with FAD that are prerequisite steps to the covalent attachment of FAD. Arg-42 and Thr-45, along with Tyr-44, comprise part of a highly conserved flavin binding sequence, RXY(T,S), that is found in other flavoproteins, several of which have well-defined X-ray crystal structures. We tested the roles of Arg-42 and Thr-45 in MAO B by constructing mutant MAO B cDNAs which encode amino acid substitutions at these residues and expressed the variant proteins in COS-7 cells. Substitution of Arg-42 or Thr-45 with alanine resulted in complete loss of MAO B activity and FAD incorporation. However, conservative substitutions of Arg-42 with lysine or Thr-45 with serine resulted in MAO B variants that retain both partial activity and partial FAD incorporation. These results indicate that Arg-42 and Thr-45 form critical noncovalent interactions with FAD that are required for the subsequent activation of MAO B by covalent coupling of FAD.

Characterization of a highly conserved FAD-binding site in human monoamine oxidase B.

Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the apoenzyme at Cys397. Our previous studies identified two noncovalent flavin-binding regions in MAO B (residues 6-34 and 39-46) (Kwan, S.-W., Lewis, D. A., Zhou, B. P., and Abell, C. W. (1995) Arch. Biochem. Biophys. 316, 385-391; Zhou, B. P., Lewis, D. A., Kwan, S.-W., Kirksey, T. J., and Abell, C. W. (1995) Biochemistry 34, 9526-9531). In these regions, Glu34 and Tyr44 were found to be required for the initial binding of FAD. By comparing sequences with enzymes in the oxidoreductase family, we now have found an additional FAD-binding site in MAO B (residues 222-227), which is highly conserved across species (human, bovine, and rat). This conserved sequence contains adjacent glycine and aspartate residues (Gly226 and Asp227). Based on the x-ray crystal structures of several oxidoreductases (Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol. 212, 135-142; Van Driessche, G., Kol, M., Chen, Z.-W., Mathews, F. S., Meyer, T. E., Bartsch, R. G., Cusanovich, M. A., and Van Beeumen, J. J. (1996) Protein Sci. 5, 1753-1764), the Gly residue at the end of a beta-strand facilitates a sharp turn and extends the beta-carbonyl group of Asp to interact with the 3'-hydroxyl group of the ribityl chain of FAD. To assess the hypothesis that Gly226 and Asp227 are involved in FAD binding in MAO B, site-specific mutants that encode substitutions at these positions were prepared and expressed in mammalian COS-7 cells. Our results indicate that Gly226 and the beta-carbonyl group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent binding of FAD. Our studies also reveal that mutagenesis at Glu34 and Tyr44 not only interferes with covalent flavinylation and catalytic activity of MAO B, but also with noncovalent binding of FAD. Based on these collective results, we propose that the coupling of FAD to the MAO B apoenzyme is a multistep process.

Monoamine oxidase B isolated from bovine liver exists as large oligomeric complexes in vitro.

The quaternary structure and subunit composition of bovine liver monoamine oxidase B (MAO B) was investigated using size-exclusion chromatography, sucrose gradient centrifugation and electron microscopy. Purified enzyme was subjected to Superdex gel filtration column chromatography in the presence of the non-ionic detergents, n-octyl beta-D-glucopyranoside (octyl glucoside) and Triton X-100R-PC (Triton). The specific activity and elution profiles indicate that the enzyme exists as a dimer and preferentially functions as larger oligomeric complexes. Distribution of the oligomeric forms of MAO B was found to be dependent upon protein concentration. Dilution of the enzyme, however, had little or no effect upon the specific activity profiles. In Triton and octyl glucoside, plots of specific activity versus molecular mass displayed a sigmoidal shape. The chromatographic data suggest that detergent-solubilized bovine liver MAO B exists as cooperative oligomeric enzyme complexes. Similarly, sucrose density gradient centrifugation of purified MAO B exhibited a direct correlation between enzyme activity and molecular mass of the MAO complexes. MAO B activity was found to be widely distributed throughout the sucrose gradient and the highest enzyme activity was contained in the high-density sucrose layers. MAO B specific activity is dependent upon the size of the protein complexes and, therefore, oligomerization of the enzyme may play a role in the regulation of MAO B. Transmission electron microscopy of purified MAO B was performed using protein prepared by octyl glucoside extraction. Purified enzyme was applied to Formvar-coated copper grids and negatively stained with methylamine tungstate. MAO-B-specific monoclonal antibody (MAO B-1C2) conjugated to colloidal gold was used as a probe. Contrast enhancement of the electron microscopy data showed that detergent-depleted enzyme tends to aggregate in a linear arrangement of oligomeric complexes. Our data suggest that the MAO B oligomeric complexes are hexamers which contain threefold rotational symmetry. The individual complexes have globular morphology and the hexamers appear to be composed of a trimer of MAO B homodimers.

Improved models for pharmacological null experiments: calculation of drug efficacy at recombinant D1A dopamine receptors stably expressed in clonal cell lines.

Modern drug discovery demands accurate knowledge of the drug properties of affinity and efficacy at specific receptor proteins. Furthermore, drugs with well defined properties make better tools with which to explore and understand receptor regulation. The use of clonal cell lines stably expressing a given recombinant receptor may provide a highly useful model in which drug effects may be studied on one receptor subtype at a time. The present report was designed to evaluate the utility of a general method in which a clonal cell line stably expressing a recombinant D1A dopamine receptor was used as a model system for studying drug actions by null models. The null model for receptor occlusion (to calculate agonist Ka) and the null model for relative efficacy (to calculate test agonist affinity and epsilon r) were evaluated in these studies. To initiate these studies, rat C6 glioma cells that do not normally express DA receptors have been modified by stable transfection with the primate D1A DA receptor [Machida et al., 1992 (Molec. Pharmacol. 41: 652-659)] to a density of approximately equal to fmol/mg protein. The recombinant receptors show robust stimulation of cAMP in the stably transfected C6 cells. Calculation of agonist Ka from dose-response data requires that a portion of the cell's receptors be occluded in the absence of changes in post-receptor events leading to the response. Receptor reserve is typically reduced by alkylation, thereby lowering maximal response. Unfortunately, most of the currently available alkylating agents are not selective either for a particular receptor or for receptors vs other proteins within a signaling pathway. Short-term agonist treatment offers a possible complement to the use of non-selective or poorly characterized alkylating drugs for reducing maximum response in appropriate cell systems. The null method of receptor occlusion was used to determine the Ka for dopamine when maximum response was decreased by alkylation vs short-term agonist treatment. Direct non-linear curve fitting was used to analyze the data. In addition to DA, two other compounds were used to reduce receptor reserve to validate the method: fenoldopam (relatively high efficacy) and SKF38393 (low efficacy). Analyses indicated that the affinity of DA was similar whether calculated by alkylation (1.1 +/- 0.58 microM), 75 min DA treatment (0.57 +/- 0.16 microM) or 45 min treatment with DA (0.86 +/- 0.11 microM). Short-term agonist treatment experiments using multiple concentrations of DA, fenoldopam, or SKF38393 to decrease receptor reserve provided additional support for the validity of the Ka determinations using this procedure. Other experiments were conducted according to the null model for relative efficacy in which the affinity for DA is calculated by comparing the DA response before and after receptor occlusion, and the affinity and relative intrinsic efficacy of the test agonist are determined as a function of its actions relative to DA. We used the following four test drugs: + Br-APB, a novel agent with potential dopamine agonist properties, and three high-affinity DA agonists, fenoldopam, R-(-)-apomorphine (APO), and SKF38393. Intrinsic efficacy values relative to that of DA (1.0) were as follows: fenoldopam, 0.46 +/- 0.11; APO, 0.19 +/- 0.13; SKF38393, 0.07 +/- 0.01; and +Br-APB, 0.26 +/- 0.40. The agonist affinities (Ka) were: fenoldopam, 0.018 +/- 0.008 microM; APO, 0.80 +/- 0.18 microM; SKF38393, 0.16 +/- 0.04 microM; BR-APB, 0.43 +/- 0.29 microM; and DA, 0.58 +/- 0.17 microM. EC50/Ka ratios were consistent with relative intrinsic efficacies and Ka values were similar to KL values reported for membrane binding studies. Finally, Monte Carlo simulations were conducted to determine the precision of the parameter estimates...

Cellular expression of mRNAs encoding monoamine oxidases A and B in the rat central nervous system.

Monoamine oxidases A and B (MAO-A and MAO-B) oxidatively deaminate neurotransmitter and xenobiotic amines. The cellular localization of these isoenzymes in the central nervous system (CNS) differs markedly and only partly reflects the distribution of their presumed natural substrates. In the present study, by using in situ hybridization with 35S-labelled oligonucleotide probes, we examined the distribution of mRNAs encoding MAO-A and MAO-B in the rat CNS. Probes for tyrosine hydroxylase, histidine decarboxylase, and tryptophan hydroxylase mRNAs were used to demonstrate the catecholaminergic, histaminergic, or serotoninergic nature of some cell populations in adjacent sections. The radioligands [3H]-Ro 41-1049 and [3H]lazabemide (reversible and selective inhibitors of MAO-A and MAO-B, respectively) were used to reveal the protein distribution by enzyme radioautography. The distribution and abundance of transcripts for both isoenzymes in the tissues investigated differed markedly but, in general, correlated with the protein distribution. MAO-A mRNA and protein were most abundant in noradrenergic neurons. However, moderate levels of transcript expression and protein were also detected in the serotoninergic neurons, and low but significant levels were detected in the dopaminergic neurons. An unexpectedly remarkable degree of hybridization signal was apparent in nonaminergic cell populations, e.g., in the cerebral cortices, the hippocampal formation (CA1-3, dentate gyrus), the cerebellar granule cell layer, and the spinal cord motoneurons. In contrast, MAO-B mRNA and protein were most abundant in serotoninergic and histaminergic neurons, Bergmann glial cells, and circumventricular organs, including the ependyma. MAO-B transcripts were also weakly expressed in nonaminergic cells, e.g., in the hippocampal formation (CA1-2). A further nonneuronal localization of MAO-B transcripts was also resolved, e.g., in the glia limitans, the olfactory nerve layer, and the cerebellar peduncle. These findings reveal further the potential of various cell populations to synthesize the isoenzymes, and homologous (aminergic) and heterologous (nonaminergic) patterns of expression as well as coexpression of MAO mRNAs are described.

Flavinylation of monoamine oxidase B.

Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the enzyme at Cys397. In this study, we developed a novel riboflavin-depleted (Rib-) COS-7 cell line to study the flavinylation of MAO B. ApoMAO B can be obtained by expressing MAO B cDNA in these cells. We found that MAO B is expressed equally in the presence or absence of FAD and that apoMAO B can be inserted into the outer mitochondrial membrane. Flavinylation of MAO B was achieved by introducing MAO B cDNA and different flavin derivatives simultaneously into Rib- COS-7 cells via electroporation. Since the addition of riboflavin, FMN, or FAD resulted in equal levels of MAO B activity, we conclude that the flavin which initially binds to apoMAO B is FAD. In our previous work, we used site-directed mutagenesis to show that Glu34 in the dinucleotide-binding motif of MAO B is essential for MAO B activity, and we postulated that this residue is involved in FAD binding. In this study, we tested the role of residue 34 in flavin binding by expressing wild-type or mutant MAO B cDNA in Rib- COS-7 cells with the addition of [14C]FAD. We found that Glu34 is essential for both FAD binding and catalytic activity. Thus, FAD binds to MAO B in a dual manner at Glu34 noncovalently and Cys397 covalently. We conclude that Glu34 is critical for the initial non-covalent binding of FAD and is instrumental in delivering FAD to the covalent attachment site at Cys397.

Mutagenesis at a highly conserved tyrosine in monoamine oxidase B affects FAD incorporation and catalytic activity.

Monoamine oxidase B (MAO B), an integral protein of the outer mitochondrial membrane, catalyzes the oxidative deamination of various neuroactive and vasoactive amines. A covalently bound FAD cofactor at Cys-397 of human MAO B is required for the oxidation of the amine substrates. In addition to the covalent binding site, MAO B also contains a noncovalent FAD binding region (residues 6-34) known as the dinucleotide binding motif. Previously, we have shown that Glu-34 is required for catalytic activity, presumably by forming a hydrogen bond between the carboxylate group of glutamate and the 2'-hydroxyl group of ribose in the AMP moiety of FAD. In this work, we have identified a third FAD binding site in MAO B (residues 39-46) by sequence comparisons to other flavoenzymes. The conserved sequence contains a tyrosine residue (Tyr-44) which, based on the X-ray crystal structure of ferredoxin-NADP+ reductase, is postulated to participate in FAD binding through van der Waals contact with the isoalloxazine ring and a hydrogen bond to the 3'-hydroxy of the ribityl moiety. To test the postulated role of this tyrosine residue, site-directed mutants that encode substitutions at Tyr-44 were prepared and expressed in mammalian COS-7 cells. Variant MAO B enzymes were then characterized with respect to enzymatic activity and [14C]FAD incorporation. Substitution of tyrosine with phenylalanine had no effect on MAO B activity or the level of [14C]FAD incorporation compared to the wild-type enzyme, indicating that the hydroxyl group of the tyrosine residue was not essential at residue 44.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a dinucleotide-binding site in monoamine oxidase B by site-directed mutagenesis.

Monoamine oxidase B (MAO B), an integral protein of the outer mitochondrial membrane, catalyzes the oxidative deamination of neuroactive and vasoactive amines. The oxidation step is coupled to the reduction of an obligatory FAD cofactor. In this study, we have examined the role of one amino acid (Glu34) in human MAO B that is thought to play a crucial role in binding to the 2'-hydroxy group of ribose in the AMP moiety of FAD. Glu34 is located within a region of the MAO B molecule of high sequence identity to the dinucleotide-binding site in other flavoproteins. In MAO B, this region is postulated to consist of a beta 1-sheet-alpha-helix-beta 2-sheet motif which culminates with a Glu at the C-terminal end of the second beta-sheet. We used site-directed mutagenesis to convert Glu at position 34 to Asp, Gln, and Ala. The wild-type and mutant cDNAs were then transiently transfected into COS-7 cells and assayed for MAO B activity. All three variants exhibited a dramatic decrease in the enzymatic activity as compared to wild-type MAO B, and only the Asp variant retained any detectable activity. Our studies indicate that the Glu34 residue in human MAO B is essential for catalysis. Whether Glu34 is responsible only for alignment of the FAD for participation in the oxidation/reduction cycle or also for the initial binding of FAD to the apoenzyme remains to be determined.

Platelet MAO activity in type I and type II alcoholism.

Lowered activity of the enzyme MAOB in the platelets and other tissues of alcoholics than of nonalcoholics is the most replicated biological finding in genetic research in alcoholism. Data presented here and elsewhere also indicate that the relationship between MAOB activity and alcoholism extends to the clinical subtypes referred to as Type I and Type II alcoholism. A detailed examination of the relationship between in vitro platelet MAOB activity levels, alcoholic subtype, and general mental health status among the relatives of the probands suggests that low MAOB activity is a marker of increased risk overall and that the families of Type II alcoholics have a higher genetic risk loading than do the families of Type I alcoholics. This increased genetic loading is probably due to the classification of Type II alcoholics on the basis of features related to severity of illness and additional psychiatric features such as personality disorders. Although the families of alcoholics tend to have higher levels of psychiatric illness compared to the general population, the overall risk is compounded in the families of Type II alcoholics, and these differences in underlying risk are reflected in the observed differences in MAOB activities. Thus, MAOB is not a biological/genetic marker of alcoholism sensu stricto but is rather a biological/genetic marker of an underlying pathophysiologic process leading to alcoholism and other psychiatric illness. The task now before us is to understand this process and how the activity of MAOB is involved.

Intracellular distribution of monoamine oxidase A in selected regions of rat and monkey brain and spinal cord.

Monoamine oxidase A and B (MAO A and B; EC 1.4.3.4) are integral proteins of the outer mitochondrial membrane that degrade monoamines including the neurotransmitters norepinephrine, dopamine, and serotonin. In this study, monoclonal antibodies that recognize rat or monkey MAO A were used in immunocytochemical studies to visualize the subcellular localization of this enzyme within neurons in the central nervous system of these species. The regions examined included the locus coeruleus, substantia nigra, spinal cord, and pallidostriatum, which are known to contain MAO A-positive structures. Ultrastructural studies revealed that most MAO A staining was associated with the outer membrane of mitochondria, within the cell bodies, dendrites, axons and terminals. However, some immunoreactive staining for MAO A was also observed in the rough endoplasmic reticulum in the cell bodies. Staining for mitochondrial MAO A in dendrites was observed in terminal fields of the monoamine system, including the spinal cord and the pallidostriatum. The intensity of staining also increased in the subsynaptic density. MAO A was also found associated with mitochondria in ependymal cells lining the fourth ventricle adjacent to the locus coeruleus and in the endothelial cells lining the blood vessels. Localization of MAO A in noradrenergic neurons, ependymal cells, and subsynaptic regions of dendrites in monoamine terminal fields supports the concept that this neurotransmitter-degrading enzyme may play a protective role in the central nervous system.

A genetic familial study of monoamine oxidase B activity and concentration in alcoholics.

Platelet monoamine oxidase B (MAO B) activity and concentration were studied in a small sample of alcoholic families (n = 8) and in 20 unrelated, nonalcoholic controls. Complex segregation analyses of familial data indicated that both activity and concentration are controlled by a single major gene locus with a multifactorial background effect accounting for 0-50% of the variance. When the alcoholic family members (n = 24) were compared with the controls, all determinations of activity display significant differences, whereas MAO B concentration levels showed no difference. These results indicated that the lowered MAO B activities frequently reported among alcoholics do not reflect a change in the number of MAO B macromolecules expressed in platelets, but could be caused by the presence of an inhibitor or by a polymorphic or variant form of the enzyme.

cDNA cloning and sequencing of rat monoamine oxidase A: comparison with the human and bovine enzymes.

1. The nucleotide and deduced amino acid sequences of rat liver MAO A were determined, and sequence identities among MAO A and B from rat, human and bovine were compared. 2. MAO A from rat exhibited greater than 85% sequence identity with bovine and human MAO A, and 70% identity with rat MAO B. 3. Rat adrenal cDNAs were restriction mapped, partially sequenced and found to be identical to rat liver MAO A, suggesting that these two tissues express the same polypeptide.

Molecular properties of monoamine oxidases A and B.

Monoamine oxidases A and B (MAO-A and B) catalyze the oxidative catabolism of biogenic amines and xenobiotics. Investigation of these mitochondrial membrane proteins shows that they differ in substrate preference, inhibitor specificity, tissue and neuronal cell distribution, immunological properties, and nucleotide and deduced amino acid sequences. Comparisons of MAO-A and B from the human, bovine, and rat species show strikingly high similarity (85-88%) in the amino acid sequences of each enzyme. Furthermore, three regions in MAO-A and B have sequence identities across species of 78, 88, and 86%. These regions correspond to a nucleotide-binding site near the N-terminal end that is found in the vast majority of enzymes that require flavin adenine dinucleotide (FAD), a region of unknown function, and the FAD-binding site toward the C-terminal end. Genomic clones of MAO-B which span almost the entire gene (greater than 40 kb) have been isolated, restriction mapped, and partially sequenced. Likewise, genomic clones of MAO-A that correspond to the 3'-flanking region have also been investigated. Current studies which focus on identification of the promoter and regulatory sequences should help to establish why MAO-A and B are localized in different subsets of neurons in brain.

Inhibition of monoamine oxidases A and B by simple isoquinoline alkaloids: racemic and optically active 1,2,3,4-tetrahydro-, 3,4-dihydro-, and fully aromatic isoquinolines.

A series of 1,2,3,4-tetrahydro-, 3,4-dihydro-, and fully aromatic isoquinolines were tested as substrates and/or inactivators of highly purified human monoamine oxidase A and B (MAO A and B). None were found to be a substrate for either enzyme, but many of these isoquinolines could selectively inhibit either MAO A or B. Stereoselective competitive inhibition of MAO A was found with the R enantiomer of all the stereoisomers tested, including salsolinol (Ki = 31 microM), salsoline (Ki = 77 microM), salsolidine (Ki = 6 microM), and carnegine (Ki = 2 microM). As a class, the 3,4-dihydro-isoquinolines were the most potent inhibitors tested (Ki = 2-130 microM), and the fully aromatic isoquinolines had intermediate activity (Ki = 17-130 microM) against MAO A. In contrast, only a few of these compounds markedly inhibited MAO B. 1,2,3,4-Tetrahydroisoquinoline, its 2-methyl derivative, and o-methylcorypalline gave apparent Ki values of 15, 1, and 29 microM, respectively, and two 3,4-dihydroisoquinolines (compounds 22 and 25) showed substantial inhibition of MAO B (Ki = 76 and 15 microM, respectively). These results support the concept that the topography of the inhibitor binding site differs in MAO A and B.

Isolation, characterization, and application of monoclonal antibodies to rat tyrosine hydroxylase.

Tyrosine hydroxylase (TH, tyrosine 3-monooxygenase; EC 1.14.16.2) activity in crude extracts of rat pheochromocytoma, rat brain, and bovine adrenal medulla can be immunoprecipitated in an indirect assay by monoclonal antibodies prepared against partially purified rat pheochromocytoma TH. One of these monoclonal antibodies, TH-2D8-2, can be used for immunocytochemical localization of TH in cell bodies, dendrites, and axons in catecholaminergic neurons (e.g., cells in the substantia nigra) of rat brain and in the cell body, neurites, and growth cones of rat pheochromocytoma cells after treatment with nerve growth factor. When linked to Affi-gel 10, this monoclonal antibody can also be used for immunoaffinity purification of rat and bovine TH. These results suggest that TH-2D8-2 is a valuable reagent with which to investigate the localization, physiological regulation, and function of this important enzyme.

The effect of debrisoquin on MAO A and MAO B activities.

To examine the mode of action of debrisoquin (DEB), we studied the effect of this drug in vitro on MAO A and MAO B enzyme activities. DEB was shown to be a competitive inhibitor of highly purified human MAO A and MAO B enzyme activities. DEB inhibited placental MAO A with a Ki value of 0.5 microM and liver MAO B with a Ki value of 8.8 microM, 18-fold greater effect on the A form. Kynuramine was used as substrate for both enzymes. Additional studies using a dilution technique showed that DEB was a reversible inhibitor of both forms of the enzyme. The results of this study show that DEB is a potent competitive and reversible inhibitor of both MAO A and MAO B enzymes.

cDNA cloning of human liver monoamine oxidase A and B: molecular basis of differences in enzymatic properties.

The monoamine oxidases play a vital role in the metabolism of biogenic amines in the central nervous system and in peripheral tissues. Using oligonucleotide probes derived from three sequenced peptide fragments, we have isolated cDNA clones that encode the A and B forms of monoamine oxidase and have determined the nucleotide sequences of these cDNAs. Comparison of the deduced amino acid sequences shows that the A and B forms have subunit molecular weights of 59,700 and 58,800, respectively, and have 70% sequence identity. Both sequences contain the pentapeptide Ser-Gly-Gly-Cys-Tyr, in which the obligatory cofactor FAD is covalently bound to cysteine. Based on differences in primary amino acid sequences and RNA gel blot analysis of mRNAs, the A and B forms of monoamine oxidase appear to be derived from separate genes.

Localization of distinct monoamine oxidase A and monoamine oxidase B cell populations in human brainstem.

Monoclonal antibodies, specific for either monoamine oxidases A or B, were used to determine the localization of monoamine oxidase in the human brain. Two distinct populations of neurons were detected by immunocytochemical staining. Neurons in regions rich in catecholamines were positive for monoamine oxidase A, including the nucleus locus coeruleus, the nucleus subcoeruleus and the medullary reticular formation. In these regions, monoamine oxidase A could be co-localized with the synthetic enzyme, dopamine-beta-hydroxylase. Neurons in the substantia nigra and the periventricular region of the hypothalamus, areas rich in dopamine neurons, stained for monoamine oxidase A but with much less frequency and intensity. The major accumulation of monoamine oxidase B-positive neurons was observed in the same regions in which monoamine oxidase B is found to co-localize with serotonin in monkey tissues, including the nucleus raphe dorsalis and the nucleus centralis superior. In addition, both monoamine oxidase A and B were localized in distinct populations of neurons in the lateral and tuberal regions of the hypothalamus, a region shown recently to contain histamine neurons in rats. Some glial cells were stained throughout the brain for monoamine oxidase A or B suggesting that glia are capable of either expression or uptake of these proteins.