Mitochondrial Monoamine Oxidase

Monoamine oxidase of bovine kidney mitochondria has been purified to high specific activity by solubilization with digitonin, ammonium sulfate fractionation, and calcium phosphate gel-cellulose column chromatography. Absorption and fluorescence spectra indicated strongly that mitochondrial monoamine oxidase is a flavoenzyme. Under anaerobic conditions, benzylamine reduced the flavin at 460 rnp apparently to 75% of the total reduction observed with sodium dithionite. The enzyme preparation contained approximately 1 mole of flavin per 100,000 g of protein. The flavin prosthetic group was not characterized precisely, due to the tight association of this group with the protein. Analysis of monoamine oxidase exhibited 7 to 8 sulfhydryl equivalents per lo5 g of protein. When varying amounts of silver nitrate or p-chloromercuribenzoate were incubated with the enzyme, the activity was observed to decrease as a linear function of the amount of inhibitor added; complete inhibition was obtained at a ratio of 7.3 moles of thiol-characterizing reagent per lo5 g of protein. Similarly, the extent of reduction of the flavoenzyme by benzylamine, at 460 mp, was proportional to the metallic reagent-enzyme ratio, indicative of a selective inhibitory process. The copper content of the purified enzyme was found by a procedure of atomic absorption spectrophotometry to be 0.14 to 0.15 pg per mg of protein. On the basis of such values and the flavin content there would be 4 or 5 eq of catalytically active flavin per atom of copper, indicating the nonessentiality of copper for activity of this oxidase. These observations are not in agreement with conclusions offered recently by other authors. In addition, there was no observable correlation between the inhibition of enzyme activity by appropriate chelating agents and the ability of these agents to chelate copper. Other interpretations for the inhibition produced by certain of these agents are proposed.