Enzyme Localization in Beef‐Heart Mitochondria

Abstract

Treatment of heavy beef heart mitochondria with different concentrations of digitonin, followed by centrifugation of the treated suspension resulted in a release of mitochondrial enzymes in a certain order. Monoamine oxidase and rotenone‐insensitive NADH cytochrome c reductase were found primarily in the outer membrane fraction. At low concentrations of digitonin adenylate kinase and creatine kinase were in part released pointing to an intracristal localization; the remainder of these enzymes was found to be associated with the inner membrane‐matrix fraction. The soluble citric cycle enzymes were found in the inner membrane‐matrix preparation. They were released from the matrix in two groups: (a) isocitrate dehydrogenase, malate dehydrogenase, fumarase and aconitase and (b) 2‐oxoglutarate dehydrogenase, pyruvate dehydrogenase and lipoate dehydrogenase. Enzymes intrinsic to the inner membrane were released at concentrations of digitonin higher than 0.4 mg/mg protein (cytochrome oxidase, succinate dehydrogenase, ATPase and 3‐hydroxybutyrate dehydrogenase). The absence of citric cycle enzyme activity, except succinate dehydrogenase in submitochondrial particles prepared by sonication, also points to a localization of these enzymes in the matrix. From the experiments with the digitonin method it is concluded that the majority of the shared mitochondrial enzymes has a similar localization both in beef heart and rat liver mitochondria. Detergents such as digitonin, Lubrol WX and Titron X‐100 induce the “twisted” configuration of the inner membrane of beef heart mitochondria, suspended in a sucrose medium, as visualized electron microscopically.