Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis

TEMPO-substituted pargyline analogues differentially inhibit recombinant human monoamine oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria, suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane [Upadhyay, A., and Edmondson, D. E. (2009) Biochemistry 48, 3928]. This approach is extended to the recombinant rat enzymes and to rat liver mitochondria. The differential specificities exhibited for human MAO A and MAO B by the m- and p-amido TEMPO pargylines are not as absolute with the rat enzymes. Similar patterns of reactivity are observed for rat MAO A and B in mitochondrial outer membrane preparations expressed in Pichia pastoris or isolated from rat liver. In intact yeast mitochondria, recombinant rat MAO B is inhibited by the pargyline analogue whereas MAO A activity shows no inhibition. Intact rat liver mitochondria exhibit an inhibition pattern opposite to that observed in yeast where MAO A is inhibited and MAO B activity is unaffected. Protease inactivation studies show specificity in that MAO A is sensitive to trypsin whereas MAO B is sensitive to β-chymotrypsin. In intact mitochondrial preparations, MAO A is readily inactivated in rat liver but not in yeast upon trypsin treatment and MAO B is readily inactivated by β-chymotrypsin in yeast but not in rat liver. These data show MAO A is oriented on the cytosolic face and MAO B is situated on the surface facing the intermembrane space of the mitochondrial outer membrane in rat liver. The differential mitochondrial outer membrane topology of MAO A and MAO B is relevant to their inhibition by drugs designed to be cardioprotectants or neuroprotectants.     

Monoamine oxidases A and B are differentially regulated by glucocorticoids and “aging” in human skin fibroblasts

Two forms of monoamine oxidase (MAO A and MAO B) exist which, although similar in a number of properties, can be distinguished on the basis of their substrate specificity, inhibitor sensitivity, kinetic parameters, and protein structure. These properties were used to study the molecular mechanism(s) by which glucocorticoid hormones and "aging," known to alter MAO activity in vivo, regulated the expression of MAO A and MAO B in cultured human skin fibroblasts. The addition of dexamethasone or hydrocortisone to cultures resulted in a dose- and time-dependent increase in total MAO activity, whereas the removal of hormone from cultures resulted in a time-dependent decrease in activity toward control levels. The response to dexamethasone was affected by culture conditions such as serum concentration, feeding frequency, and cellular "age." Cellular aging, in the absence of hormone, also resulted in increased levels of total MAO activity. The effects of hormones and aging on total MAO activity appeared to be selective for MAO A. The 6- to 14-fold increases in total activity were paralleled by similar increases in the activity and amount of active MAO A but less than 2- to 3-fold increases in the activity and amount of MAO B. Altered synthesis or degradation of the active enzyme appeared to account for the effects of hormones, aging, and various culture conditions on MAO activity. Inhibitor sensitivity, substrate affinity, electrophoretic mobility, and molecular turnover number of either form of the enzyme were not altered during dexamethasone treatment or during cellular aging. However, rates of active MAO synthesis were affected by hormone treatment and feeding frequency, rates of active MAO degradation by serum concentration, and rates of active MAO synthesis or degradation by aging. In summary, we have shown that glucocorticoids and cellular aging selectively affect the amount of MAO A at the level of active enzyme synthesis or degradation. Further, our finding that the expression of the two forms of MAO in human fibroblasts can be independently regulated supports the growing evidence that MAO A and MAO B are separate molecular entities.     

2-Naphthylamine, a compound found in cigarette smoke, decreases both monoamine oxidase A and B catalytic activity

Cigarette smokers exhibit a lower monoamine oxidase (MAO; EC 1.4.3.4) activity than nonsmokers. MAO is located in the outer membrane of mitochondria and exists as two isoenzymes, MAO A and B. MAO A prefers 5-hydroxytryptamine (serotonin), and MAO B prefers phenylethylamine (PEA) as substrate. Dopamine is a substrate for both forms. 2-Naphthylamine is a carcinogen found in high concentrations in cigarette smoke. The results of this study show that 2-naphthylamine has the ability to inhibit mouse brain MAO A and B in vitro by mixed type inhibition (competitive and non-competitive). The Ki for MAO A was determined to be 52.0 microM and for MAO B 40.2 microM. The inhibitory effect of 2-naphthylamine on both MAO A and B catalytic activity, supports the hypothesis that smoking decreases MAO activity in vivo, instead that smokers with lower MAO activity are more prone to become a smoker.     

Phe(208) and Ile(199) in human monoamine oxidase A and B do not determine substrate and inhibitor specificities as in rat

It has been reported previously that reciprocally switching Phe(208) and Ile(199) in rat monoamine oxidase (MAO) A and B, respectively, was sufficient to switch their substrate and inhibitor preferences. In this study, the same mutants were made in the human forms of MAO. When compared with MAO A, MAO A-F208I showed a sixfold decrease in the specificity constant k(cat)/K(m) for both the MAO A- and the MAO B-preferring substrates 5-hydroxytryptamine and beta-phenylethylamine, respectively. The reciprocal point mutant MAO B-I199F had no effect on substrate affinity. To investigate if the region neighboring these two residues is responsible for conferring preferences, we have also made chimeric constructs by reciprocally switching the corresponding amino acid segments 159-214 in MAO A and 150-205 in MAO B. Chimerics MAO AB(159-214)A and MAO BA(150-205)B had small changes in K(m) and IC(50) values when compared with MAO A and B, respectively, but did not exhibit a preference switch. The results suggest that Phe(208) in MAO A and amino acid segments 159-214 and 150-205 in MAO A and B, respectively, influence the enzyme active site. However, substrate and inhibitor preferences of human MAO A and B are not determined by the respective residues Phe(108) and Ile(199) as in rat MAO nor by their neighboring regions.     

Influence of flavin analogue structure on the catalytic activities and flavinylation reactions of recombinant human liver monoamine oxidases A and B.

Two riboflavin-deficient (rib5) Saccharomyces cerevisiae expression systems have been developed to investigate the influence of riboflavin structural alterations on the covalent flavinylation reaction and activity of recombinant human liver monoamine oxidases A and B (MAO A and B). Nineteen different riboflavin analogues were tested with MAO A and nine with MAO B. MAO expression and flavinylation were determined immunochemically with antisera to MAO and an anti-flavin antisera. Expression levels of both MAO A and B are invariant with the presence or absence of riboflavin or riboflavin analogues in the growth medium. Flavin analogues with a variety of seven and eight substitutions are found to be covalently incorporated and to confer catalytic activity. The selectivities of MAO A and MAO B for flavin analogue incorporation are found to be similar, although 8alpha-methylation of the flavin resulted in a higher level of catalytic activity for MAO B than for MAO A. N(3)-Methylriboflavin and 8-nor-8-aminoriboflavin are not covalently bound as they are not converted to their respective FAD forms by yeast. 5-Carba-5-deazaflavin and 7,8-nor-7-chlororiboflavin are not covalently incorporated into MAO A and do not support catalytic activity. A flavin peptide was isolated from MAO A containing 7-nor-7-bromo-FAD and was demonstrated to be covalently attached to Cys-406 by an 8alpha-S-thioether linkage by sequence analysis and by matrix-assisted laser desorption ionization time of flight mass spectroscopy. MAO A partially purified from yeast grown on 8-nor-8-chlororiboflavin exhibited an absorption spectrum indicating the covalent flavin is an 8-nor-8-S-thioflavin, suggesting a nucleophilic displacement mechanism that supports the quinone-methide mechanism previously suggested as a general mechanism for covalent flavin attachment.     

Molecular characterization of monoamine oxidase in zebrafish (Danio rerio)

Monoamine oxidase (MAO) is responsible for the degradation of a number of neurotransmitters and other biogenic amines. In terrestrial vertebrates, two forms of the enzyme, named MAO A and B, were found in which mammals are coded by two similar but distinct genes. In teleosts, the biochemical data obtained so far indicate that enzyme activity is due to a single form, whose sequence, obtained for trout, displays 70% identity with mammal MAO A and B. In this paper, we carried out an investigation of zebrafish MAO (Z-MAO) to shed further light on the nature of the MAO form present in aquatic vertebrates. Sequencing studies have revealed an open reading frame 522-amino-acids long with MW 58.7 kDa, displaying 84% identity with trout MAO and about 70% identity with mammal MAO A and MAO B. Analysis of the sequence and of the predicted secondary structure shows that also in Z-MAO principal domains characterizing the MAOs are present. The domain linking the FAD is very well conserved, while the transmembrane domain sequence linking the enzyme to the external mitochondrial membrane does not appear to be conserved even with respect to trout MAO. Comparison with the amino acids which, according to the human MAO B and rat MAO A models, line the substrate-binding site shows that in Z-MAO, several residues (V172, N173, F200, L327) differ from MAO B but are similar or identical to the corresponding ones present in rat MAO A, as well as in trout MAO. A three-dimensional model is reported of the substrate-binding site of Z-MAO obtained by comparative modeling. Our observations support the hypothesis that the MAO form present in aquatic vertebrates is a MAO A-like form. Experiments performed to test the effect of selective MAO A (clorgyline) and MAO B (deprenyl) inhibitors on the enzyme's activity in liver and brain confirm the presence of a single form of MAO in zebrafish.     

Monoamine oxidase of rat skeletal muscle: substrate specificity and half-life

The substrate specificity of rat skeletal muscle MAO has been studied. By the use of clorgyline as a MAO A inhibitor, it is found that 5-hydroxytryptamine, tryptamine, and kynuramine are deaminated by MAO A whereas benzylamine is a substrate for both forms of MAO. Phenethylamine displays a concentration-dependent preference for the two forms of MAO. These substrate specificies of the two forms of MAO in skeletal muscle are different from those observed in liver and brain but resemble closely that seen with heart. The half-lives of MAO A and MAO B in muscle estimated by rate of recovery from pargyline inhibition are 6.9 and 6.4 days, respectively.     

Inactivation of monoamine oxidase A by the monoamine oxidase B inactivators 1-phenylcyclopropylamine, 1-benzylcyclopropylamine, and N-cyclopropyl-alpha-methylbenzylamine

Three known mechanism-based inactivators of beef liver mitochondrial monoamine oxidase (MAO) B are tested as inactivators of human placental mitochondrial MAO A. 1-Phenylcyclopropylamine (1-PCPA), 1-benzylcyclopropylamine (1-BCPA), and N-cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) are time-dependent irreversible inactivators of MAO A. The KI values for 1-PCPA and N-C alpha MBA, analogues of the MAO B substrate benzylamine, are much higher with MAO A than with MAO B. Evidence is presented to show that 1-PCPA inactivates MAO A by attachment to the flavin cofactor, unlike the reaction with MAO B in which 1-PCPA can attach to both a cysteine residue and the flavin [Silverman, R.B., & Zieske, P.A. (1985) Biochemistry 24, 2128-2138]. The reaction of 1-BCPA with MAO A was too slow to study in detail. N-C alpha MBA exhibits the same properties toward inactivation of MAO A that it does for inactivation of MAO B. Attachment in both cases is shown to be to one cysteine residue per enzyme molecule. The results with 1-PCPA indicate that the active site topographies of MAO A and MAO B are different. The ability of N-C alpha MBA to undergo attachment to a cysteine residue in both MAO A and MAO B may lead the way toward peptide mapping of the two isozymes in order to determine differences in their primary structures.     

Structural comparison of human monoamine oxidases A and B: mass spectrometry monitoring of cysteine reactivities

Monoamine oxidases (MAO) A and B are approximately 60-kDa outer mitochondrial membrane flavoenzymes catalyzing the degradation of neurotransmitters and xenobiotic arylalkyl amines. Despite 70% identity of their amino acid sequences, both enzymes exhibit strikingly different properties when exposed to thiol-modifying reagents. Human MAO A and MAO B each contain 9 cysteine residues (7 in conserved sequence locations). MAO A is inactivated by N-ethylmaleimide (NEM) much faster (tau(1/2) = approximately 3 min) than MAO B (tau(1/2) = approximately 8 h). These differences in thiol reactivities are also demonstrated by monitoring the NEM modification stoichiometries by electrospray mass spectrometry. Inactivation of either enzyme with acetylenic inhibitors results in alterations of their thiol reactivities. Cys5 and Cys266 were identified as the only residues modified by biotin-derivatized NEM in clorgyline-inactivated MAO A and pargyline-inactivated MAO B, respectively. The x-ray structure of MAO B (Binda, C., Newton-Vinson, P., Hubalek, F., Edmondson, D. E., and Mattevi, A. (2002) Nat. Struct. Biol. 9, 22-26) shows that Cys5 is located on the surface of the molecule opposite to the membrane-binding region. Cys266 in MAO A is predicted to be located in the same region of the molecule. These thiol residues are also modified by biotin-derivatized NEM in the mitochondrial membrane-bound MAO A and MAO B. This study shows that the MAO A structure is "more flexible" than that of MAO B and that clorgyline and pargyline inactivation of MAO A and B, respectively, increases the structural stability of both enzymes. No evidence is found for the presence of disulfide bonds in either enzyme, contrary to a previous suggestion.     

Monoamine Oxidases A and B as Components of a Membrane Complex

Abstract: The activities of monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B) represent two independent types of substrate binding site, as indicated by experiments with selective inhibitors and also by substrate competition. We have tried to determine whether A and B active sites of human brain and liver MAO are located on physically separable enzyme forms or as subunits in large membrane-bound complexes. MAO was extracted from several sources by a procedure that was designed to give solubilized enzyme in high-speed supernatants, with ratios of MAO A/MAO B activities similar to those in initial crude homogenates. This solubilized enzyme gave gel filtration profiles that suggested the presence of large molecular complexes. Affinity binding experiments indicated that both MAO A and B activities may occur on the same complexes in tissues that initially contain both activities. These complexes were broken down to enzymatically active subunits by treatment with either low concentrations of sodium dodecyl sulfate, with phospholipase A₂, or with a combination of both agents. Results of this study support a concept of MAO as part of a membrane unit in which A and B are two distinct enzymes embedded in a phospholipid structure. The enzymatic activity of MAO A is critically dependent on associated phospholipids, whereas that of MAO B is not.     

Monoclonal antibodies to monoamine oxidase B and another mitochondrial protein from human liver.

A monoclonal antibody has been generated to human liver monoamine oxidase (MAO) B by fusion of mouse myeloma cells with spleen cells from a mouse immunized with a mixture of semi-purified MAO A and MAO B. The antibody, 3F12/G10, an immunoglobulin G1, reacts with its antigen in cryostat sections of human liver, showing an intracellular particulate distribution as demonstrated by immunoperoxidase staining. The antibody indirectly precipitates [3H]pargyline-labelled human MAO B both from liver and platelet extracts but fails to precipitate MAO A from liver extracts. The antibody does not recognise rat liver MAO B, showing that the determinant is not universally expressed on MAO B. The antibody has no effect on the catalytic activity of MAO B. Other monoclonal antibodies were generated but they are directed to a protein with a subunit Mr of 54 000, a contaminant of the MAO preparation. One of these antibodies, A8/C2, an IgG2a, reacts with the same protein in both rat and human liver extracts.     

MONOAMINE OXIDASE (EC 1.4.3.4): ISOLATION AND CHARACTERIZATION OF MULTIPLE FORMS OF THE BRAIN ENZYME

Monoamine oxidase (MAO) in crude mitochondrial preparations from rat brain was solubilized, and different MAO-active fractions were separated by agarose columns and by Sephadex electrophoresis. Any combination of these techniques yielded at least three fractions possessing MAO activity as measured by assays using radioactive serotonin and benzylamine as substrates. The molecular weight of one of the MAO forms was found to be approximately 400,000 daltons while another was at least 1.5 × 10⁶ daltons. The crude mitochondria1 MAO was inhibited by [¹⁴C]-labelled pargyline and then solubilized and the radioactivity of the soluble and particulate MAO was compared to the enzyme activity found in the soluble and particulate fractions. Our studies suggest that appreciable MAO activity is lost upon solubilization and that the conformation of MAO may be altered.     

Monoamine oxidase and catechol-O-methyl transferase activity in Tetrahymena.

Tetrahymena pyriformis strain HSM was found to have monomine oxidase (MAO) and a catechol-3-methyl transferase-like (COMT) activity. As in mammalian tissues, the MAO activity is predominantly localized in the mitochondrial pellet and COMT in the cytosol. The COMT-like activity was present in amounts comparable to several mouse tissues and was inhibited by tropolone. MAO activity was much lower than in any of the mouse tissues tested, and its activity varied greatly from preparation to preparation. The substrate preference of Tetrahymena MAO was tryptamine greater than serotonin greater than dopamine, and activity increased with increasing pH from pH 6.5 to pH 7.8, as does that of mouse liver MAO. Teh Km of Tetrahymena MAO for tryptamine was approximately 4 micrometer, an order of magnitude lower than that of mouse liver MAO. Sensitivity of inhibition by MAO inhibitors was variable. In some preparations, no inhibition was observed. In others clear inhibition was obtained, harmine and clorgyline being among the most potent inhibitors.     

Localization of monoamine oxidases A and B in primate brains relative to neuron-specific and non-neuronal enolases

Using serotonin and phenylethylamine deamination as measures of MAO A and MAO B activity respectively, positive correlations were observed between the activities of MAO A and MAO B in different areas of rhesus monkey and human brains. When the activities of MAO A and MAO B were compared with those of neuron-specific enolase and nonneuronal enolase (isozymes which are markers for neurons and glia), a slight but non-significant correlation was observed, suggesting that a simple distribution of MAO A in neurons and MAO B in glia is unlikely. This conclusion is supported by studies using synaptosomes, but contrasts with that from investigations of MAO from peripheral tissues, where experiments indicate that MAO A is predominantly localized in neurones.     

Exploring the structural basis of the selective inhibition of monoamine oxidase A by dicarbonitrile aminoheterocycles: Role of Asn181 and Ile335 validated by spectroscopic and computational studies

Since cyanide potentiates the inhibitory activity of several monoamine oxidase (MAO) inhibitors, a series of carbonitrile-containing aminoheterocycles was examined to explore the role of nitriles in determining the inhibitory activity against MAO. Dicarbonitrile aminofurans were found to be potent, selective inhibitors against MAO A. The origin of the MAO A selectivity was identified by combining spectroscopic and computational methods. Spectroscopic changes induced in MAO A by mono- and dicarbonitrile inhibitors were different, providing experimental evidence for distinct binding modes to the enzyme. Similar differences were also found between the binding of dicarbonitrile compounds to MAO A and to MAO B. Stabilization of the flavin anionic semiquinone by monocarbonitrile compounds, but destabilization by dicarbonitriles, provided further support to the distinct binding modes of these compounds and their interaction with the flavin ring. Molecular modeling studies supported the role played by the nitrile and amino groups in anchoring the inhibitor to the binding cavity. In particular, the results highlight the role of Asn181 and Ile335 in assisting the interaction of the nitrile-containing aminofuran ring. The network of interactions afforded by the specific attachment of these functional groups provides useful guidelines for the design of selective, reversible MAO A inhibitors.     

Steroid regulation of monoamine oxidase activity in the adrenal medulla

Administration of different steroid hormones in vivo has distinct and specific effects on the MAO activity of the adrenal medulla. In an effort to reconstitute these effects in defined cells, we have isolated endothelial cells and chromaffin cells from the bovine adrenal medulla and tested each cell type for sensitivity to these steroids. As in the intact animal, we found that endothelial cell MAO activity was stimulated 1.5- 2.5-fold by 10 microM progesterone, hydrocortisone, and dexamethasone, inhibited by ca. 50% by 17-alpha-estradiol, but unaffected by testosterone. The type of MAO in the endothelial cells was found to be exclusively of the A type. The chromaffin cells had MAO B exclusively and were inert to treatment with dexamethasone. The mode of action of the various steroids on MAO A activity in endothelial cells seemed to be that of affecting the number of MAO molecules, as binding of [3H]pargyline, an MAO inhibitor, changed in proportion to changes in enzyme activity. Consistently, the kinetic parameters for MAO A showed changes in Vmax but not Km under all conditions. The specificity of steroid action on MAO A activity was also supported by the fact that steroid-induced changes in total cell division ([14C]thymidine incorporation) and total protein synthesis ([14C]leucine incorporation) were seen after changes in MAO A. We conclude that the differential effects of steroids on MAO activity in the intact adrenal medulla can be reproduced in cultured adrenal medullary endothelial cells but not in chromaffin cells. Therefore we suggest that the action of these steroid hormones on the intact adrenal medulla may be restricted to the endothelial cell component of this tissue.     

Substrate and inhibitor specificities for human monoamine oxidase A and B are influenced by a single amino acid

Monoamine oxidase (MAO) is responsible for the oxidation of biogenic and dietary amines. It exists as two isoforms, A and B, which have a 70% amino acid identity and different substrate and inhibitor specificities. This study reports the identification of residues responsible for conferring this specificity in human MAO A and B. Using site-directed mutagenesis we reciprocally interchanged three pairs of corresponding nonconserved amino acids within the central portion of human MAO. Mutant MAO A-I335Y became like MAO B, which exhibits a higher preference for beta-phenylethylamine than for the MAO A preferred substrate serotonin (5-hydroxytryptamine), and became more sensitive to deprenyl (MAO B-specific inhibitor) than to clorgyline (MAO A-specific inhibitor). The reciprocal mutant MAO B-Y326I exhibited an increased preference for 5-hydroxytryptamine, a decreased preference for beta-phenylethylamine, and, similar to MAO A, was more sensitive to clorgyline than to deprenyl. These mutants also showed a distinct shift in sensitivity for the MAO A- and B-selective inhibitors Ro 41-1049 and Ro 16-6491. Mutant pair MAO A-T245I and MAO B-I236T and mutant pair MAO A-D328G and MAO B-G319D reduced catalytic activity but did not alter specificity. Our results indicate that Ile-335 in MAO A and Tyr-326 in MAO B play a critical role in determining substrate and inhibitor specificities in human MAO A and B.     

Blood-Brain Barrier Monoamine Oxidase: Enzyme Characterization in Cerebral Microvessels and Other Tissues from Six Mammalian Species, Including Human

We studied the enzyme monoamine oxidase (MAO) in isolated cerebral microvessels, and in mitochondria-enriched brain and liver preparations from six mammalian species, including human. We also studied MAO distribution in various tissues and in discrete brain regions of the rat. MAO was assessed by measuring the specific binding of [3H]pargyline, an irreversible MAO inhibitor, and the rates of oxidation of known MAO substrates: benzylamine, tyramine, tryptamine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Molecular forms of MAO were examined by using specific MAO inhibitors, and by polyacrylamide gel electrophoresis after [3H]pargyline binding. In general, the liver from all species had higher MAO levels than the brain, with minor variation among species in their brain and liver MAO content. However, there were remarkable species differences in brain microvessel MAO, with rat microvessels having one of the highest MAO activity among all tissues, whereas MAO activities in brain microvessels from humans, mice, and guinea pigs were very low. In most rat tissues, including the brain, there was a preponderance of MAO-B over MAO-A. The only exceptions were the heart and skeletal muscle. Estimates of MAO half-life in rat brain microvessels, rat brain, and rat liver indicated that microvessel MAO had a higher turnover rate. The reasons underlying the remarkable enrichment of rat cerebral microvessels with MAO-B are unknown, but it is evident that there are marked species differences in brain capillary endothelium MAO activity. The biological significance of these findings vis a vis the role of MAO as a "biochemical blood-brain barrier" that protects the brain from circulating neurotoxins and biogenic amines should be investigated.