Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation

Abstract Silicotungstate was found to remove the inner membrane spheres from submitochondrial particles derived from bovine heart. Both particulate and soluble ATPase were shown to be rapidly inactivated in the presence of low concentrations of silicotungstate. A simple and reproducible procedure for the preparation of deficient submitochondrial particles by treatment with silicotungstate was developed. These particles were resolved with respect to all known coupling factors as well as with respect to succinate dehydrogenase. Addition of reconstitutively active succinate dehydrogenase together with F1, F2, F3, and F5 restored oxidative phosphorylation with succinate as substrate. Addition of a succinate dehydrogenase preparation was required also for reconstitution of phosphorylation associated with the oxidation of DPNH or 32Pi-ATP exchange activity. This effect is due to the presence of a new coupling factor in the preparation.