Preparation and Some Properties of Sub-Mitochondrial, Reduced Diphosphopyridine Nucleotide Oxidizing Particles from Isolated Mitochondria.

Abstract

A simple method for preparation and isolation of sub-mitochondrial particles from isolated rat or guinea-pig liver mito- chondria is described. It consists of grinding the mitochondria with Al2O3 in a mortar and subsequent differential centrifugation. Some proper-ties of afraction, which is centrifuged down at 100,000 g (60 min centrifuga-tion), are described. The fraction oxidizes reduced diphosphopyridine nucleotide at a much higher rate than reduced triphosphopyridine nucleotide, both with oxygen and with cytochrome c as electron acceptors. The oxidation of succinate with cytochrome c is strongly stimulated by phenazine methosulfate. Antimycin A inhibits the oxidation of succinate in the absence of phenazine methosulfate, which can mediate electron transport from succinate to cytochrome c around the antimycin A-sensitive site. When a catalytic amount of diphosphopyridine nucleotide is added, [beta]-hydroxybutyrate is oxidized by added cytochrome c in the presence of the sub-mitochrondrial fraction whereas glutamate oxidation occurs only when also the final supernatant is added. This indicates that mitochondrial dehydrogenases are differently distributed in the sub-mitochondrial fractions.