Studies on Mitochondrial Proteins. II. Localization of Components in the inner membrane labelling with diazobenzenesulfonate, a non-penetrating probe

Abstract

The topography of the inner membrane of rat liver mitochondria was studied using a probe, diazobenzenesulfonate, which interacts preferentially with surface components. Inner membranes were examined both in a native orientation as found in the intact mitochondrion or in an inverted state as found in isolated inner membranes prepared by sonication. Enzyme inactivation as a consequence of diazobenzenesulfonate labeling was employed to determine the localization of a number of inner membrane activities. In inner membranes labeled on the outer surface, NADH and succinate oxidation were strongly inhibited while ATPase and ascorbate-N,N,N′,N′- tetramethyl-p- phenylene-diamine (TMPD) oxidase activities were unaffected. In inner membranes labeled on the inner surface. ATPase and succinate oxidation were inactivated while NADH oxidation and ascorbate-TMPD oxidase were unaffected. Succinate dehydrogenase was inhibited only by labeling the inner surface while NADH dehydrogenase was inhibited to a similar extent by treatment of either surface. Sodium dodecylsulfate-polypeptides (66 000 and 26 000) on the outer surface of the inner membrane and five polypeptides (80 000, 66 000, 51 000-48 000, and 26 000) on the inner surface. These results indicate a highly asymmetric localization of inner membrane components.