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 Km values ranged from 1.1 ± 10−5m for tryptamine to 2.5 ± 10−4m for dopamine; (2) substrate specificity from Vmax 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.

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Abstracl~ 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 sub->(rate. The deamination of the biogenic amines: serotonin. dopamine, tyramine, tryptamine, phenylethylilminc and two non-physiological amines, kynuramine and rn-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 monovdent 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 M A 0 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 M A 0 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, values obtained at pH 7.4, 8.2 and 9.1 were corrected for ionization of the substrate, the corrected K , was lowest at pH 7.4 for all substrates.Less than 50'" of M A 0 activity was lost when beef brain mitochondria was heated at 5 0 C for 20 min. However. there was only a slight variation with substrate in the thermal inactivation experiments. It is concluded that the mitochondria1 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.

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