Characteristics of mitochondrial and synaptosomal monoamine oxidase in monkey brain

Enzymic properties of monoamine oxidase (MAO) from monkey brain were studied. High MAO activity was observed in the mesencephalon and dienecephalon of the brain. Highest activity in every region of the brain was found with tyramine as a substrate. Monkey brain mitochondrial MAO showed a different substrate specificity and different Km and Vmax values than the enzyme from mice, rats, guinea pigs and rabbits. The pH activity curves were all bell-shaped, but the pH optima were remarkably different with the various substrates used. The activities of various substrates at pH 7.2 were compared with those at the pH optimum. At the pH optima, the activity was about 1.2-fold higher with tyramine and dopamine, 2-fold higher with beta-phenylethylamine (beta-PEA) and 3-fold higher with serotonin (5-HT) and benzylamine. These results were almost similar when synaptosomes from monkey brain were used. MAO activities with 5-HT and beta-PEA were strongly inhibited by much lower concentrations of clorgyline and deprenyl, respectively. Plateau-shaped inhibition curves by these inhibitors were obtained with tyramine as the substrate. These results indicate that both the A- and B-form of MAO appear to be uniformly distributed in monkey brain, and the A-form of MAO represents approximately 35% and 50% of the total MAO activity in mitochondria and synaptosomes, respectively.     

Amine competition for oxidation by rat liver mitochondrial monoamine oxidase

Mixed substrate experiments have been carried out with rat liver mitochondrial monoamine oxidase. The results of these studies are interpreted in terms of the known specificities and sensitivities to inhibition of the two kinetically distinguishable species present in this preparation. The results of this study are discussed in terms of the function of the enzyme in vivo.     

The effect of tris buffers on rat liver mitochondrial monoamine oxidase

The effect of tris buffers upon the monoamine oxidase (MAO) activity in rat liver mitochondria has been investigated. Tris buffer was shown to inhibit MAO in a non-competitive manner with a Ki of 15–25 mm. Tyramine, 5-hydroxytryptamine and β-phenethylamine but not benzylamine oxidations were all inhibited by tris buffer. All inhibitions, except that of 5-HT, were completely reversible. It is suggested that these effects are produced by conformational changes in the structure of the MAO. The significance of these results is discussed with respect to the use of tris buffers in the extraction and estimation of the activity of MAO.     

Endogenous monoamine oxidase inhibitor-like substances in monkey brain

The extraction and partial purification of endogenous "monoamine oxidase (MAO) inhibitor-like" material from the monkey brain are described. The endogenous material (F-1 and F-2) obtained after Bio-Gel P-2 gel filtration and silica column chromatography inhibited MAO in the monkey brain mitochondria toward 5-hydroxytryptamine (5-HT), beta-phenylethylamine (beta-PEA), tyramine and dopamine as substrates. The inhibitory effects of F-1 and F-2 were non-linear concentration dependent, and F-1 non-competitively inhibited A-form MAO, while F-2 inhibited A-form MAO competitively and inhibited B-form MAO non-competitively. These substances were more potent inhibitors of A-form than of B-form MAO. F-2 was heat stable but liable to the treatment with pepsin and trypsin. F-1 was not inactivated by heat treatment and digestion with pepsin and trypsin. F-1 may be a low molecular weight (less than 1350) compound, including certain monoamines or their metabolites or other unidentified compounds, while F-2 was a low molecular weight (about 2500) peptide.     

Styrene inhibits monoamine oxidase A, but not monoamine oxidase B in monkey brain mitochondria

The effects of styrene on mitochondrial monoamine oxidase (MAO) activity in rat and monkey brains were compared in vitro. After preincubation at 25 degrees C for 20 min with 1 mM styrene monomer MAO-A activity in monkey brain was inhibited potently using 5-HT (for MAO-A substrate), but MAO-B activity in monkey brain and platelets were slightly inhibited using beta-PEA (for MAO-B substrate). Styrene monomer also competitively inhibited MAO-A activity in a dose-dependent manner. MAO-A in monkey brain was inhibited by styrene in ascending order of potency: styrene trimer>styrene dimer>styrene monomer. In contrast styrene monomer slightly inhibited both MAO-A and MAO-B activities in rat brain mitochondria. In the present study styrene monomer potently inhibits MAO-A activity, but not MAO-B activity, in monkey brain mitochondria in vitro. These results indicate the inhibiting action of styrene differs depending on animal species and MAO isoforms.     

The effect of tris buffers on rat liver mitochondrial monoamine oxidase.

The effect of tris buffers upon the monoamine oxidase (MAO) activity in rat liver mitochondria has been investigated. Tris buffer was shown to inhibit MAO in a non-competitive manner with a Ki of 15-25 mM. Tyramine, 5-hydroxytryptamine and beta-phenethylamine but not benzylamine oxidations were all inhibited by tris buffer. All inhibitions, except that of 5-HT, were completely reversible. It is suggested that these effects are produced by conformational changes in the structure of the MAO. The significance of these results is discussed with respect to the use of tris buffers in the extraction and estimation of the activity of MAO.     

Some interrelated properties of A and B form monoamine oxidase in monkey brain mitochondria

The multiplicity of monoamine oxidase (MAO) in monkey brain was studied by comparing the relationship between the selective substrates of MAO and the pH-activity curves obtained using these substrates. When mitochondrial and A-form MAO were used as the enzyme preparations with serotonin (5-HT) and norepinephrine (NE), preferential substrates for A-form MAO, the pH optima were 8.8 and 7.8 with 5-HT and 8.5 and 7.2 with NE. These substrates were also oxidized by B-form MAO after changing the pH of the incubation medium (shift to alkaline); these pH optima were 9.0 and 8.2, respectively. When common substrates of MAO were used (tyramine, octopamine, dopamine and tryptamine), the pH activity curves obtained were all broad and bell-shaped with pH optima for the 3 species of enzyme (mitochondria, A-form and B-form MAO) at 8.0, 7.8, and 8.0 with tyramine; 8.3, 7.5, and 8.5 with octopamine; 7.8, 7.5, and 8.5 with dopamine; and 8.0, 8.3, and 6.9 with tryptamine, respectively. The pH optima were 6.6 with beta-phenylethylamine (beta-PEA) and 9.0 with benzylamine, preferential substrates for B-form MAO, for either mitochondria or B-form MAO. The Km values obtained for tryptamine and beta-PEA were lower than those for the other substrates of MAO, regardless of the enzyme preparations. The Km and Vmax values of both forms MAO for 5-HT and NE were similar to those of the A-form MAO. The differences in the Km and Vmax values of the A-form MAO and B-form MAO for common substrates were comparable. Tyramine, octopamine and dopamine were substrates for both forms MAO, with only a slight preference for B-form MAO over A-form MAO. However, tryptamine may be deaminated predominantly by A-form MAO.     

Purification and some properties of porcine brain mitochondrial monoamine oxidase B.

Monoamine oxidase was isolated from pig brain mitochondria in a highly purified state by the following procedures: treatment with Ca-phosphate gel, column chromatography on DEAE-cellulose, mercurial-Sepharose, and hydroxyapatite chromatography and gel filtration on Bio-Gel A-1.5m. The properties of the enzyme disclosed that it is a monoamine oxidase of the B-type. The FAD present in the enzyme was covalently attached to the protein and it was shown to react with S-N,N-dimethylamino-1-propyne which is indicative of a reaction with the N-⁵ position of the isoalloxazine ring of FAD. The flavin content from spectral data and from the ¹⁴C-content of the enzyme inactivated with labeled 3-N,N-dimethylamino-1-propyne yielded a FAD content of 1 mole/104,000 g of protein. The subunit molecular weight from SDS-disc electrophoresis yielded a molecular weight of 52,000. Thus, it was concluded that the brain monoamine oxidase probably has two identical subunits, only one of which contained covalently bonded FAD.     

Amethocaine-induced inhibition of mitochondrial monoamine oxidase activity

Amethocaine (tetracaine) (1-10 microM) produces a concentration-dependent in-vitro inhibition of mitochondrial membrane-bound MAO activity towards tyramine (18-84% in brain and 19-84% in liver) and 5-hydroxytryptamine (5-HT) (23-94% in brain and 20-100% in liver). At relatively higher concentrations (25-300 microM) of amethocaine, benzylamine oxidation is inhibited in brain (24-91%) and liver (29-100%). The extent of MAO inhibition is appreciably reduced when preincubation time of the enzyme with a low concentration (7.5 microM) of amethocaine is increased from zero to 45 min. This inhibition is reversible. The Km of MAO for tyramine is increased in brain (106-473%) and liver (121-352%) in the presence of amethocaine (2-7.5 microM) accompanied by a decrease in Vmax (21-51% in brain and 18-57% in liver). Similarly the Km of MAO for 5-HT is increased to the extent of 79-336% in brain and 51-225% in liver and the corresponding Vmax is decreased by 35-55% and 39-74%, respectively, in the presence of 2-5 microM amethocaine. At relatively higher concentrations (25-100 microM), amethocaine increases the Km of MAO for benzylamine in brain (25-101%) and liver (26-85%) and decreases the Vmax by 28-64% and 32-63% in the respective tissues. Thus these results suggest that amethocaine preferentially inhibits MAO-A and the nature of inhibition is reversible and of mixed type.     

Isoelectric focusing of isoenzymes of monkey platelet monoamine oxidase

Monkey platelet monoamine oxidase (MAO) was preferentially found as the B-form of the enzyme as observed from differences in substrate specificities, as well as liver MAO. The isoelectric points and molecular weights of platelet MAO subunits were compared with those of monkey liver using sodium dodecyl sulfate-disc polyacrylamide gel electrophoresis and isoelectric focusing-disc gel electrophoresis. The pI value of monkey liver was a single peak at 6.5, but the pI values of monkey platelets were triple peaks at 5.5, 6.5 and 7.0. The molecular weight of MAO subunits in monkey platelets was similar to that of liver, and was found to be about 60,000. These results indicate that MAO-B of monkey platelets differs from MAO-B of the liver, and that it has different electrophoretic properties.