Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β-d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K_m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.     

Enzymologic characteristics of monoamine oxidase from liver of the skipjack tuna Katsuwonus pelamis

Substrate and inhibitory specificity of mitochondrial monoamine oxidase (MAO) from liver of skipjack tuna Katsuwonus pelamis was studied. The results of substrate—inhibitory analysis with application of chlorgilin and deprenyl might be indirect proofs of existence of one molecular MAO form in the tuna liver. Studied enzyme, as liver MAO of terrestrial mammals, deaminates tyramine, tryptamine, dopamine, serotonin, noradrenalin, benzylamine, β-phenylethylamine, N-methylhistamine and does not deaminate histamine, is not suppressed by 10 mM semicarbazide. Takrin, acriflavin, proflavin, acridine orange and pyronine G were established to be irreversible inhibitors of middle strength in respect to MAO of tuna liver. The specificity of inhibitors action upon deamination of various substrates was equal.     

Comparative enzymologic study of catalytical properties of liver monoamine oxidases of sturgeon fish

We carried out the comparative study of the substrate and inhibitory specificity of liver monoamine oxidases (MAO) of the giant sturgeon Huso huso, the starred sturgeon Acipenser stellatus, the Persian sturgeon Acipenser persicus, and the Russian sturgeon Acipenser gueldenstaedtii. Results of the substrate-inhibitor analysis with use of inhibitors chlorgilin and deprenil, as well as five specific substrates indicate homogeneity of these enzymes. All studied MAO have the several orders higher sensitivity to chlorgilin than to deprenil, with essential interspecies differences being observed. There are determined kinetic parameters of enzymatic deamination (K _M and V) of tyramine, serotonin, noradrenalin, benzylamine, β-phenylethylamine, and N-methylhistamine. All studied enzymes have been established to have the higher activity toward serotonin and noradrenalin-substrates of the MAO A form as compared with benzylamine, β-phenylethylamine, and N-methylhistamine-substrate of the mammalian MAO B form, the maximal activity being characteristic of the giant sturgeon.     

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.     

Comparative study of catalytic properties of mink and rat liver monoamine oxidase

Comparative substrate-inhibitor analysis of catalytic properties of mitochondrial monoamine oxidase (MAO) of liver of the American mink Mustela vison Schreber and of liver of Wistar rat has been performed. It has been found that MAO of mink, like MAO of rat, has properties of classic mammalian MAO: it deaminates tyramine, tryptamine, serotonin, benzylamine, β-phenylethylamine and does not deaminate histamine as well as does not have sensitivity to semicarbazide. Study of kinetics of the monoamine oxidase deamination revealed both qualitative and quantitative differences between these enzymes. Specificity of action on MAO-A form of four irreversible inhibitors—acridine derivatives—has been shown; this specificity was several times higher for the mink liver MAO than for the rat liver MAO. It is suggested that the liver MAO of both species of the studied animals has several isoenzyme forms or several centers of the substrate binding.     

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.     

Monoamine oxidase activity measurements using radioactive substrates

The use of Amberlite CG-50, Dowex 50 and solvent extraction for separation of the oxidation products of the biogenic amines are compared, and measurements of monoamine oxidase activity using ¹⁴C-labeled biogenic amines are described. K_m data for tyramine, dopamine, tryptamine, and serotonin for monoamine oxidase activity of rabbit brain mitochondria are reported. Rates of product formation from [¹⁴C]tyramine are compared with polarographic measurements of oxygen utilization using purified MAO and intact mitochondria from rabbit liver and brain. Difficulties in comparative measurements of monoamine oxidase activity and some reasons for wide variations in published data are discussed.     

Some parameters affecting the activity of monoamine oxidase in purified bovine brain mitochondria.

Abstract— Some parameters affecting the activity of monoamine oxidase (MAO) in purified beef brain mitochondria were investigated, and diversities in enzyme properties were found as a function of substrate. The deamination of the biogenic amines: serotonin, dopamine, tyramine, tryptamine, phenylethylamine and two non-physiological amines, kynuramine and m-iodobenzylamine, was studied. Anions in high concentrations inhibited enzyme activity with kynuramine being the substrate most affected. Among the biogenic amines, the activity with the indolalkylamines showed greater sensitivity to mono-valent anions such as chloride than to polyvalent ions such as phosphate whereas the opposite was true with the phenylalkylamines. However, pyrophosphate ion had little or no effect on MAO activity, regardless of substrate. The inhibition of kynuramine and serotonin deamination was non-competitive but mixed competitive inhibition was found with tyramine and phenylethylamine. The activity of MAO was markedly affected by pH, and it had been previously reported that the substrates showed different pH optima in their oxidation. The effect of pH on activity has been attributed in part to changes in the ionization of the substrate and the hypothesis that the true substrate is the non-protonated amine. This was reflected in kinetic studies showing high substrate inhibition with increased pH. It was calculated that phenylethylamine would have the highest percentage of un-ionized amine at pH 8.2 and 9.1. At these pHs, there was more pronounced inhibition with high substrate concentrations of phenylethylamine than with the other substrates. In contrast, there was little inhibition with high substrate concentrations of tyramine which was the most ionizable of the substrates tested. When K_m values obtained at pH 7.4, 8.2 and 9.1 were corrected for ionization of the substrate, the corrected K_m was lowest at pH 7.4 for all substrates. Less than 50% of MAO activity was lost when beef brain mitochondria was heated at 50°C for 20 min. However, there was only a slight variation with substrate in the thermal inactivation experiments. It is concluded that the mitochondrial membrane environment surrounding the enzyme imposes certain restrictions on the enzymatic activity with respect to the different substrates which, in turn, are also affected by such parameters as pH and ions. The results are discussed in terms of the relationship of these factors to the question of enzyme multiplicity.     

Substrate-inhibitory analysis of monoamine oxidase from hepatopancreas of the octopus <Emphasis Type="Italic">Bathypolypus arcticus</Emphasis>

Study of the substrate-inhibitory specificity of mitochondrial monoamine oxidase (MAO) of hepatopancreas of the octopus Bathypolypus arcticus revealed distinctive peculiarities of catalytic properties of this enzyme. The studied enzyme, on one hand, like the classic MAO of homoiothermal animals, is able to deaminate tyramine, serotonin, benzylamine, tryptamine, b-phenylethylamine, while, on the other hand, it deaminates histamine and does not deaminate putrescine-classic substrates of diamine oxidase (DAO). Results of the substrate-inhibitory analysis with use of chlorgiline and deprenyl are indirect proofs for the existence in the octopus hepatopancreas of one molecular MAO form. Semicarbazide and pyronine G turned out to be weak irreversible inhibitors, four derivatives of acridine-irreversible inhibitors of the intermediate effectiveness with respect to the octopus hepatopancreas MAO; specificity of action of inhibitors at deamination of different substrates was equal.     

Studies of monoamine oxidases: properties of the enzyme in bovine and rabbit brain mitochondria

Brain mitochondria were prepared from rabbit and bovine cerebral cortex and the purity and intactness of the preparation assessed through the use of enzyme markers and electron microscopy. Enzymatic properties of monoamine oxidase were studied in the purified mitochondrial preparations which were essentially devoid of major contamination by other organelles, especially microsomes. Five substrates were used for characterization of the enzyme: dopamine, kynuramine, serotonin, tryptamine and tyramine. It was found that there was considerable substrate variation in the properties, but in general, the two species showed similar characteristics. The more pertinent findings were: (1) apparent K_m values ranged from 1.1 ± 10^?5m for tryptamine to 2.5 ± 10^?4m for dopamine; (2) substrate specificity from V_max values in decreasing order was tyramine > dopamine > kynuramine > serotonin > tryptamine for the bovine enzyme and tyramine > kynuramine > dopamine > serotonin > tryptamine for rabbit; (3) there appeared to be three distinct pH optima according to substrate: pH 7.5 for phenylethylamines, pH 8.2–8.5 for the indolylamines and pH 9.1 for kynuramine; and (4) the activity with tyramine was highly sensitive to increased oxygen tension while kynuramine showed no sensitivity. It is proposed that the properties of monoamine oxidase, a membrane-bound enzyme, might be influenced by the microenvironment and results are also discussed in terms of multiple forms or multiple activity sites on a single form.     

Purification and characterisation of monoamine oxidase from Avena sativa

FAD-containing monoamine oxidase (MAO; EC 1.4.3.4) oxidises monoamines to their corresponding aldehydes, H₂O₂, and NH₃. It has been purified to homogeneity in mammals, but to our knowledge, there have been no reports of the enzyme in plants. MAO activity was detected in Avena sativa seedlings during germination using benzylamine as substrate. The enzyme was purified to homogeneity (as assessed by native PAGE) by Sephadex G-25, DEAE Sephacel, hydroxyapatite, Mono Q, and TSK-GEL column chromatographies. The molecular mass estimated by gel filtration using the TSK-GEL column was 220?kDa. SDS-PAGE yielded four distinct protein bands of 78, 58, 55, and 32?kDa molecular masses. The pI value of the enzyme was 6.3. The enzyme showed high substrate specificity for an endogenous amine, phenethylamine, which was oxidised to phenylacetaldehde, but not for ethylamine, propylamine, butylamine, pentylamine, dopamine, serotonin, tryptamine, or tyramine. The K _m values for benzylamine and phenethylamine were 2.7?×?10^?4 and 7.1?×?10^?4?M, respectively. Enzyme activity was not inhibited by pargyline, clorgyline, semicarbazide, or Na-diethyldithiocarbamate. Benzaldehyde, the product of benzylamine oxidation, exhibited strong competitive inhibition of enzyme activity with a Ki of 3???M. FAD was identified by ODS-column chromatography as an enzyme cofactor. The enzyme contained 2?mol of FAD per 220,000?g of enzyme.     

Chlorphentermine inhibits pulmonary mitochondrial monoamine oxidase

Oxidation of six amine substrates by rat, rabbit and guinea-pig lung mitochondrial monoamine oxidase (MAO) was investigated polarographically with a Clark oxygen electrode in the presence of chlorphentermine (CP). This amphiphilic drug decreased the deamination of serotonin, norepinephrine, tyramine and dopamine significantly in all three species. However, the oxidation of tryptamine and benzylamine was unchanged. Amine oxidation by MAO in guinea-pig lung mitochondria was much more sensitive to the CP-mediated inhibition than rat or rabbit. A kinetic study of serotonin oxidation in the absence and presence of CP showed that both Vmax and Km were affected. These combined data indicate that CP is a specific inhibitor of pulmonary, mitochondrial monoamine oxidase form A with mixed-type inhibition.     

Monoamine Oxidase Activities in Catfish (Parasilurus Asotus) Tissues

Abstract

The substrate- and inhibitor-related characteristics of monoamine oxidase (MAO) were studied for catfish brain and liver. The kinetic constants for MAO in both tissues were determined using 5-hydroxytryptamine (5-HT), tyramine and β-phenylethylamine (PEA) as substrates. For both tissues, the V_max values were highest with 5-HT and lowest with PEA. The K_m value for the brain was highest with 5-HT, followed by tyramine and PEA; but for the liver its value was highest with PEA, followed by 5-HT and tyramine, although all values were in the same order of magnitude. The inhibition of MAO by clorgyline and deprenyl by use of 5-HT, tyramine and PEA as substrates showed that the MAO-A inhibitor clorgyline was more effective than the MAO-B inhibitor deprenyl for both catfish tissues; a single form was present since inhibition by clorgyline or deprenyl with 1000 μM PEA showed single phase sigmoid curves. It is concluded that catfish brain and liver contain a single form of MAO, relatively similar to mammalian MAO-A.     

Comparative substrate-inhibitor analysis of mink liver monoamine oxidases

Comparative substrate-inhibitor analysis of catalytic properties of liver monoamine oxidases (MAO) was performed in the mature males of the American mink Mustela vison and the European mink Mustela lutreola. The action on the MAO activity was studied of alkaloids of the benzo[c]phenanthridine group: sanguinarine and chelidonine, diisoquinoline alkaloid berberine, medicinal agents “Ukrain” and “Sanguirythrin” as well as derivatives of 2-propylamine: deprenyl and chlorgylin. The latter turned out to be irreversible inhibitor of the MAO A form, whereas deprenyl-irreversible inhibitor of the MAO B form in both studied mink species. The selectivity of action of each inhibitor on the corresponding liver MAO form for the species M. vison was one order of magnitude stronger than for the species M. lutreola. All studied alkaloids as well medicinal agents on their basis have been shown to be specific irreversible inhibitors of the intermediate strength of the liver MAO A form of both mink species. They inhibit the enzymatic deamination of serotonin, tyramine, and tryptamine without affecting the deamination reaction of benzylamine and β-phenylethylamine (at concentrations of 10 mM and lower). Out of five studied isoquinoline agents, the medication “Ukrain” and alkaloid chelidonine have the highest inhibitory action; the agent “Sanguirythrin” and alkaloids berberine and sanguinarine produce the weaker monoamine oxidase effect. The revealed specificity of action of the studied inhibitors is an indirect evidence for the presence in the liver enzymes of both mink species, like in the rat liver enzyme, of several molecular forms.     

Comparative study of substrate and inhibitory specificity of monoamine oxidase of squid optic ganglia

Comparative study of substrate specificity of monoamine oxidase (MAO) of optic ganglia of the Pacific squid Todarodes pacificus and the Commander squid Berryteuthis magister has been carried out. The enzyme of the Pacific squid, unlike that of the Commander squid, has been established to be able to deaminate not only tyramine, tryptamine, serotonin, benzylamine, and β-phenylethylamine, but also histamine-substrate of diamine oxidase (DAO). In relation to all studied substrates, the MAO activity of optic ganglia of T. pacificus is several times higher as compared with that of B. magister. In the case of deamination of serotonin this difference was the highest and amounted to 5 times. Semicarbazide, the classic DAO inhibitor, at a concentration of 10 mM did not inhibit catalytic activity of both studied enzymes. The substrate-inhibitory analysis with use of deprenyl and clorgyline, specific inhibitors of different MAO forms, indicates homogeneity of the enzyme of the Pacific squid and heterogeneity of the Commander squid enzyme whose composition seems to contain at least two MAO forms. There are obtained quantitative differences in substrate specificity and reaction capability with respect to the inhibitors clorgylin and deprenyl for MAO of optic ganglia of the studied squid species. These differences probably can be explained by significant differences in the evolutionary level of these biological species.     

Monoamine Oxidase and Catechol-O-Methyl Transferase Activity in Tetrahymena*

SYNOPSIS Tetrahymena pyriformis strain HSM was found to have monoamine oxidase (MAO) and a catechol-O-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 > serotonin > dopamine, and activity increased with increasing pH from pH 6.5 to pH 7.8, as does that of mouse liver MAO. The K_m of Tetrahymena MAO for tryptamine was 4 μM, an order of magnitude lower than that of mouse liver MAO. Sensitivity to 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.     

Monoamine oxidase inhibition by pyrrole quinoline derivatives

The effect of 1-(beta-aminoethyl)-3H-pyrrole[2,3-h]quinoline (I), 3-(beta-aminoethyl)-1H-pyrrole[2,3-h]quinoline (I'), 8-amino-3H-pyrrole[2,3-h]quinoline (II), 6-amino-3H-pyrrole[2,3-h]quinoline (II') and 8-amino-1H-pyrrole[2,3-h]quinoline (III) on tyramine, serotonin and 2-phenylethylamine deaminase activities of mitochondrial monoamine oxidase from bovine brain were studied. All the compounds tested appeared to be reversibly inhibit MAO without preliminary incubation. Compounds II, II' and III specifically inhibited type A MAO; compound III exhibited the highest selectivity. The inhibition was of a mixed type. The effects of compounds I and I' were competitive and inconsistent with a classical concept on the dual activity of MAO, i. e., deamination of tyramine, a substrate common for MAO type A and MAO type B was inhibited in a greater degree than the deamination of specific substrates of MAO type A (serotonin) or type B (2-phenylethylamine). Possible reasons for the observed phenomenon are discussed.     

Catalytical properties of liver monoamine oxidases of the Commander squid <Emphasis Type="Italic">Berryteuthis magister</Emphasis> from various habitation zones

There has been performed kinetic analysis of enzymatic reactions of deamination of tyramine, tryptamine, serotonin, benzylamine, β-phenylethylamine, and histamine under action of liver monoamine oxidase (MAO) of the Commander squid Berryteuthis magister from various habitation zones in the Bering and Japan Seas. A substrate inhibition by high concentrations of all studied substrates has been revealed, which seems to indicate mutual effect of various MAO forms present in liver of the studied squids. Analysis of kinetic parameters of enzymatic reactions of deamination of six studied substrates and the substrate-inhibitory analysis with use of two derivatives of acridine and deprenyl indicate the enzyme heterogeneity, the presence of at least two MAO-A forms, and the absence of intraspecies differences in MAO of the Commander squids from various habitation zones. The most active was the MAO form responsible for serotonin deamination. There were obtained quantitative difference in substrate specificity and reaction ability with respect to inhibitor of proflavin for the liver MAO of the Commander and Pacific squids.     

Activation of platelet monoamine oxidase by plasma in the human.

A pronounced activation of platelet monoamine oxidase (MAO) by human plasma has been observed. The activation was substrate selective, since serotonin, $\text{p}$-tyramine, dopamine and benzylamine were much more effective than β-phenylethylamine or tryptamine. The activator(s) in the plasma was heat stable but labile to acid hydrolysis and treatment with lipase and protease. The plasma was also found to be capable of activating partially purified MAO obtained from rat liver mitochondria. Phospholipids such as phosphatidylethanolamine were shown to activate MAO.     

Species differences in lung mitochondrial monoamine oxidase activities

Pulmonary mitochondrial monoamine oxidase (MAO) activity was examined in preparations from rat, rabbit and guinea-pig with 12 different amines as substrates: serotonin, norepinephrine, and octopamine (type A specific); tryptamine, benzylamine, 5-methoxytryptamine, 5-methyltryptamine, p-methoxyphenylethylamine, and 3,4-dimethoxyphenethylamine (type B specific); and tyramine, dopamine and 3-methoxytyramine (type A + B specific). The oxidation of type A and type A + B substrates was greater in guinea-pig lung mitochondria than in rat or rabbit preparations. Except for benzylamine, the oxidation of type B substrates was similar in all three species. Benzylamine was not oxidized by guinea-pig lung mitochondria but was actively metabolized by rat and rabbit preparations.