MULTIPLE BINDING SITES OF HUMAN BRAIN MONOAMINE OXIDASE AS INDICATED BY SUBSTRATE COMPETITION

Abstract— Six endogenous substrates of monoamine oxidase (EC 1.4.3.4) (serotonin, l -norepinephrine, dopamine, tyramine, tryptamine and β -phenethylamine) were used separately and in pairs with human brain mitochondrial extracts. Apparent K ₁ values were obtained from experiments in which only 1 of 2 substrates was isotopically labelled, and these values were compared with experimental K _m values. β -Phenethylamine appears to be metabolized at enzyme active sites independent from those which bind serotonin. The substrate l -norepinephrine competes with serotonin for an enzyme site, but also may be catalysed at an additional site which is independent of serotonin binding. Experiments in which [¹⁴C]tryptamine was combined with [³E]serotonin indicated that tryptamine is a much more potent inhibitor of serotonin oxidation than was predicted from K _m values. It is suggested that the competition among substrates of MA0 which is observed in uitro may have relevance to in uiuo mechanisms for control of biogenic amine concentrations.     

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.     

Multiple catalytic sites of rat brain mitochondrial monoamine oxidase

We compared the inhibitory and catalytic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed lower K_m values for tryptamine and β-phenylethylamine than for tyramine and serotonin. The K_m values of the untreated preparation for tyramine, tryptamine, and β-phenylethylamine obtained were the same as those of the form B enzyme and the K_m value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and β-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of β-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of β-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.     

Monoamine oxidase: To B or not to B?

That the enzyme, monoamine oxidase (E.C. 1.4.3.4. amine: O₂ oxidoreductase, MAO) exists in multiple forms was first suggested by Johnston (1) who studied the effects of the irreversible inhibitor clorgyline on MAO. It has been proposed that MAO can be classified into two types, A and B, according to their inhibitor sensitivity and substrate specificity. Type A MAO was found to be solely responsible for the deamination of 5-hydroxytryptamine (5-HT) and shows high sensitivity to clorgyline, while type B MAO metabolizes 2-phenethylamine (PEA) and benzylamine (BA) and is less sensitive to clorgyline. Subsequently, it was shown that type B MAO is highly sensitive to the irreversible inhibitor deprenyl (2).Recently, the “multiple forms” concept has been questioned (3–5) mainly because of increasing evidence which is contradictory to some earlier findings. As an alternative, another hypothesis was put forward insinuating that MAO is an enzyme with multiple binding sites but only one molecular entity (3,4,6,7). This account will focus on some experimental findings accumulated mainly since 1978 and which, although equivocal, strongly support the “one molecular entity” hypothesis of MAO.     

MONOAMINE OXIDASE A AND B IN CULTURED CELLS

Abstract— Monoamine oxidase (MAO) activity against tryptamine was compared in a number of continuous rodent lines, including neuroblastoma, hepatoma, melanoma, nephroma, sarcoma and L cells. Activities against tryptamine varied over 300-fold in homogenates of different lines, being highest in hepatoma line MH₁C₁ and lowest in a neuroblastoma line lacking hypoxanthine phosphoribosyltransferase (HPRT) activity. The amount, but not the type, of MAO activity varied with the stage of growth in homogenates of neuroblastoma and hepatoma cells. Measurements of succinate-cytochrome c reductase (SCCR), another mitochondrial enzyme, also showed 20-fold variations between lines, being highest in neuroblastoma line N1E-115 and lowest in hepatoma line MH₁C₁; SCCR and MAO activities appeared to be regulated independently. The relative proportions of the A and B types of MAO activity were determined in homogenates and living cultures. Clorgyline inhibition of tryptamine deamination in homogenates indicated that in all lines except MH₁C₁, greater than 95% of the MAO activity was of the A type. In MH₁C₁ homogenates, using clorgyline or deprenyl, 40–70% of the activity appeared to be of the A type and 30-60% of the B type. In cultures of neuroblastoma N1E-115 cells, deamination of tryptamine and dopamine was sensitive to inhibition by low concentrations of clorgyline, indicating that the A type of activity is present intracellularly. as in homogenates. In MH₁C₁ hepatoma cultures, tryptamine deamination showed a biphasic sensitivity to clorgyline. We interpret this to mean that A and B types of MAO activity occur together in living hepatoma cells.     

Possible mechanism of selective inhibition of rat liver mitochondrial monoamine oxidase by chlorgiline and deprenyl]

The inhibition of the deamination of serotonin (the main substrate of monoamine oxidase (MAO) type A) by chlorgiline and deprenyl and of beta-phenylethylamine (the main substrate of the B type MAO) by fragments of rat liver mitochondrial membrane as well as the influence of 4-ethylpyridine on this process were studied. It was shown that the MAO activity of the mitochondrial membrane fragments was highly sensitive to chlorgiline, when serotonin was used as substrate, whereas a high sensitivity toward deprenyl was observed with beta-phenylethylamine as substrate. 4-Ethylpyridine (5.10(-3) M), a competitive and reversible inhibitor of the MAO activity, inhibited deamination of serotonin and beta-phenylethylamine by 34 and 30%, respectively. In experiments with chlorgiline (the specific inhibitor of MAO type A) 4-ethylpyridine (5.10(-3) M) introduced into the samples after preincubation of mitochondria with increasing concentrations of chlorgiline (30 min, 23 degrees C) decreased the inhibition by chlorgiline of the deamination of beta-phenylethylamine, but sharply increased the inhibitory effect of chlorgiline on the oxidation of serotonin. In analogous experiments with deprenyl (the specific inhibitor of MAO type B) 4-ethylpyridine (5.10(-3) M) decreased the inhibitory effect of deprenyl not only on the deamination of serotonin (substrate of MAO A), but also on the oxidation of beta-phenylethylamine (the main substrate of MAO type B). The decrease in the inhibitory effect of deprenyl on the deamination of beta-phenylethylamine after the addition of 4-ethylpyridine may be intensified upon preincubation of deprenyl with mitochondria in the presence of 4-ethylpyridine. The data obtained demonstrate the difference in the type and mechanism of inhibition of the deamination of serotonin by chlorgiline as well as in the type and mechanism of oxidation of beta-phenylethylamine by deprenyl. The possible mechanism of selective blocking of MAO activity by chlorgiline and deprenyl was discussed in terms of our previous data on the existence in the active center of mitochondrial MAO of specific sites for substrate binding, differing in their structure-functional characteristics.     

Oxidation of β-Phenylethylamine by Both Types of Monoamine Oxidase: Examination of Enzymes in Brain and Liver Mitochondria of Eight Species

Abstract: β-Phenylethylamine (PEA) was characterized as a substrate for type A and type B monoamine oxidase (MAO) in brain and liver mitochondria of eight species at different substrate concentrations. In all species, at 10.0 μM, PEA was almost specific for type B MAO. At 1000 μM, however, the amine was common for both types of MAO in rat brain and liver, human brain and liver, mouse brain, guinea pig brain and liver, and bovine brain, while it was specific for type B MAO in mouse liver, rabbit brain and liver, bovine liver, pig brain and liver, and chicken brain and liver. From the present study, when PEA is used as a type B substrate, it is recommended that the substrate concentration should be sufficiently low to avoid the effects of species and tissue differences.     

[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A—I. PROPERTIES OF THE ACTIVE SITE OF MAO A FROM RAT AND BOVINE BRAINS

A high-affinity (K_d= 5.9 nM) specific binding site for [³H]harmaline was detected in membranes from rat and bovine brains. Studies of the regional and subcellular distributions of this binding indicated its close association with monoamine oxidase type A activity (MAO A) measured with [³H]serotonin ([³H]5-HT) as the substrate. Maximal binding capacity and MAO A activity were found in mitochondrial enriched fractions. Mitochondria of synaptosomal or extra-synaptosomal origin exhibited very similar properties with respect to [³H]harmaline binding characteristics and MAO A activity. Among psychoactive drugs, only monoamine oxidase inhibitors (MAO I) prevented the specific binding of [₃H]harmaline. Logit-log inhibition curves of binding by MAO I gave only one slope which was not significantly different from 1.0, suggesting the existence of only 1 category of specific sites for [³H]harmaline in the membrane preparations from rat and bovine brains. Consistent with the preferential inhibition of MAO A by harmaline, other MAO I of this class, i.e. clorgyline and Lilly 51641, were 10²-2 × 10³ times more efficient than deprenyl and pargyline, two inhibitors of MAO type B, in displacing [³H]harmaline from its specific binding site. K_i and IC₅₀ values for the inhibition of [³H]harmaline binding by MAO I and MAO substrates (tryptamine, 5-HT, norepinephrine) were almost identical with those characterizing their action on MAO A activity with [³H]5-HT as the substrate. In conclusion, the specific binding site for [³H]harmaline exhibited all the expected properties of the active site of MAO A. Like the technique of precipitation with a specific antibody, binding of [³H]harmaline should be of great help for studying the structural characteristics of the active site of MAO A and determining the number of MAO molecules in tissues under various physiological conditions.     

THE EFFECTS OF SOLUBILIZATION PROCEDURES ON THE RELEASE AND MOLECULAR STATE OF ACETYLCHOLINESTERASE FROM ELECTRIC ORGAN TISSUE

Abstract— A study was made of the effect of various solubilization procedures on the release of AChE from electric organ tissue of the electric eel and on the molecular state of the enzyme. The procedures employed included homogenization in different ionic media or in the presence of detergents, etuymic treatment and chemical modification. Studies were performed on intact electroplax, tissue homogenates and membrane fractions. The apparent AChE activity of intact cells, homogenates and membrane fractions was shown to be governed by diffusion-controlled substrate and hydrogen ion gradients, generated by AChE-catalyscd hydrolysis, leading to a lower substrate concentration and a lower pH in the vicinity of the particulate enzyme.
Treatment of homogenates with NaCl solutions or with NaCl solutions containing the nonionic detergent Triton X-100 causes release of the native'molecular forms of the enzyme (primarily the 18 S species) which aggregate at low ionic strength. For optimal extraction both high ionic strength (e.g. 1 M-NaCl) and the detergent are needed AChE is also solubilized by treatment of tissue homogenates with trypsin, bacterial protease or collagenase. The first two enzymes caused its release as an 11 S non-aggregating form, while collagenase also produces a minor non-aggregating - 16 S component. Treatment of tissue homogenates with maleic anhydride causes release of AChE as a non-aggregating 18 S species. On the basis of the solubilization experiments it is concluded that the interaction of AChE with the excitable membrane is primarily electrostatic. The possible orientation of the enzyme within the synaptic gap is discussed.     

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 activity in embryos of pike (Esox lucius)

1. Mitochondrial MAO specific activity was measured in eggs and early embryos of the teleostean fish Esox lucius using tryptamine, 5-hydroxytryptamine (5-HT) and phenylethylamine (PEA) as substrates. 2. Tryptamine is the most readily deaminated substrate in mitochondria isolated from unfertilized eggs and embryos at the stages of cleavage, blastula and gastrula. 3. Monoamine oxidase activity gradually decreases during development and at the gastrula stage it is respectively 80% (tryptamine), 70% (5-HT) and 50% (PEA) of that found in the egg using the corresponding substrate. 4. The inhibition of egg MAO activity by clorgyline and deprenyl measured in E. lucius eggs using tryptamine as substrate, indicates the presence of a single form of MAO not corresponding to the MAO A and MAO B described in terrestrial vertebrates.     

THE CHARACTERIZATIO OF MONOAMINE OXIDASE IN CULTURED MAMMALIAN CELLS

The expression of monoamine oxidase (MAO) in a variety of mammalian cells has been investigated employing tryptamine as substrate. The enzyme present in those cell lines having sufficient activity for detailed analysis exhibited a monophasic response to the inhibitor clorgyline. On this basis the cell lines examined were found to express only A, or only B, type activity. Hypoxanthineguanine phosphoribosyl transferase (HGPRT) deficient derivatives of both MAO type A, or MAOtype B, expressing cells were examined. The HGPRT status of the cells appeared to have little influence on the expression of the enzyme.     

Effect of triton X-100 on the activity and solubilization of rat liver mitochondrial phosphatidylserine decarboxylase

It was shown that, among ionic and nonionic detergents tested, only Triton X-100 was able to stimulate the activity of rat liver phosphatidylserine decarboxylase, whereas other detergents were without effect or were inhibitory. The solubilization procedure of phosphatidylserine decarboxylase from mitochondrial membranes with Triton X-100 was elaborated. The dependence of the solubilized decarboxylase on the Triton X-100 to phosphatidylserine ratio and the inhibitory effect of Triton X-100 at its molar ratio to phospholipid higher than 5.6 was observed. No divalent cation requirement and no dependence of the ionic strength for the solubilized enzyme were observed. Kinetic parameters were determined.     

Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine.

The non-ionic detergent octyl glucoside solubilizes a substantial amount of Streptococcus faecalis membrane protein without loss of the monitored enzyme activities. A secondary detergent, dioctanoyl phophatidycholine, appears to increase the yield of solubilized material. In addition, the effect of ionic strength indicates that it may be possible to selectively extract groups of membrane proteins by their characteristic solubility at different ionic strengths. The solubilized membrane-associated enzymes, ATPase and NADH dehydrogenase, enter polyacrylamide gels as distict species. Electrophoretic studies suggest that there are two membrane-associated ATPase in the Streptococcus faecalis, one which dissociates from the membrane in the absence of Mg-2+ ions and the other which remains particulate until solubilized by detergents. Octyl glucoside can be easily removed from a solution containing solubilized proteins and lipid by dialysis.     

Monoamine Oxidase Activity in the Hepatopancreas of the Kamchatka Crab <Emphasis Type="Italic">Paralithodes camtschaticus</Emphasis>: a Substrate–Inhibitor Specificity

A study of substrate–inhibitor specificity of mitochondrial monoamine oxidase (MAO) in the hepatopancreas of the adult Kamchatka crab Paralithodes camtschaticus revealed specific catalytic properties of the enzyme. On the one hand, crab hepatopancreas MAO, like its classical hepatic counterpart, can deaminate tyramine, tryptamine, dopamine, serotonin, noradrenalin, benzylamine, β-phenylethylamine and N-methylhistamine but shows no sensitivity to 10 mM semicarbazide. On the other hand, MAO deaminates histamine but not putrescine, two classical diamine oxidase (DAO) substrates. It was established that MAO activity was several times higher toward benzylamine, β-phenylethylamine and N-methylhistamine than toward serotonin and noradrenalin. MAO was also found to be almost 500 times more sensitive to its selective inhibitor deprenyl than to chlorogilyn. A substrate–inhibitory analysis with the use of deprenyl and chloroginyl provides an indirect evidence for the existence of a sole MAO molecular form in the Kamchatka crab hepatopancreas.     

Substrate Selectivity of Type A and Type B Monoamine Oxidase in Rat Brain

Abstract: Use of the irreversible inhibitors clorgyline and deprenyl showed that rat brain mitochondria contain type A and type B monoamine oxidase (MAO). Tyramine is a substrate for both types of MAO, whereas serotonin is a preferential substrate for type A MAO. In contrast to MAO in other tissues, type A MAO in brain tissue oxidizes β-phenylethylamine (PEA) at high concentrations (0.5 and 1.0 mM). The proportions of type A and type B MAO activities in the mitochondria estimated from the double-sigmoidal inhibition curves of tyramine oxidation were about 70:30 irrespective of the concentration of tyramine. With PEA as substrate, the ratios of type A to type B activities were found to increase from low values at low concentrations to about 1 at 0.5-1.0 mM-PEA, and even higher at further increased concentrations of PEA. At very low (0.01 mM) and high (10.0 mM) concentrations of PEA, single-sigmoidal curves were obtained; with the high PEA concentration the activity was highly sensitive to clorgyline, whereas with the low concentration it was highly sensitive to deprenyl. In deprenyl-pretreated mitochondrial preparations, all the remaining activity towards 0.5-1.0 mM-PEA was shown to be highly sensitive to clorgyline, demonstrating that this activity was indeed due to oxidation by type A MAO. The opposite result was obtained with deprenyl as inhibitor of clorgyline-pretreated preparations, demonstrating that PEA at this concentration was also oxidized by type B MAO in rat brain mitochondria. The K₃ values of type A and type B MAO for PEA were significantly different. On Lineweaver-Burk analysis, plots with PEA as substrate for type A MAO in a deprenyl-treated preparation were linear over a wide concentration range, whereas those for type B MAO in a clorgyline-treated preparation were not linear, but showed substrate inhibition at higher concentrations of the substrate. It is concluded from the present findings that the effect of the substrate concentration must be considered in studies on the characteristics of multiple forms of MAO in various organs and species.     

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.     

Analysis of conserved active site residues in monoamine oxidase A and B and their three-dimensional molecular modeling

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of serotonin, norepinephrine, dopamine, and phenylethylamine. It is an outer membrane mitochondrial enzyme existing in two isoforms, A and B. We have recently generated 14 site-directed mutants of human MAO A and B, and we found that four key amino acids, Lys-305, Trp-397, Tyr-407, and Tyr-444, in MAO A and their corresponding amino acids in MAO B, Lys-296, Trp-388, Tyr-398, and Tyr-435, play important roles in MAO catalytic activity. Based on the polyamine oxidase three-dimensional crystal structure, it is suggested that Lys-305, Trp-397, and Tyr-407 in MAO A and Lys-296, Trp-388, and Tyr-398 in MAO B may be involved in the non-covalent binding to FAD. Tyr-407 and Tyr-444 in MAO A (Tyr-398 and Tyr-435 in MAO B) may form an aromatic sandwich that stabilizes the substrate binding. Asp-132 in MAO A (Asp-123 in MAO B) located at the entrance of the U-shaped substrate-binding site has no effect on MAO A nor MAO B catalytic activity. The similar impact of analogous mutants in MAO A and MAO B suggests that these amino acids have the same function in both isoenzymes. Three-dimensional modeling of MAO A and B using polyamine oxidase as template suggests that the overall tertiary structure and the active sites of MAO A and B may be similar.     

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.     

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.