AN ELECTRON-TRANSPORT SYSTEM ASSOCIATED WITH THE OUTER MEMBRANE OF LIVER MITOCHONDRIA

TLDR

Preparations of rat‑liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide in a reaction insensitive to respiratory chain inhibitors and uncoupled from phosphorylation. A swelling–shrinking, sonication, and density‑gradient centrifugation protocol separates two particulate subfractions, one rich in respiratory chain components and the other in the rotenone‑insensitive NADH‑cytochrome c reductase system. Tritiated NADH experiments show that the rotenone‑insensitive, cytochrome c‑dependent NADH oxidation pathway in rat‑liver mitochondria is distinct from the respiratory chain, is not due to microsomal contamination, and localizes to outer‑membrane derivatives that contain catalytic components similar to microsomal NADH‑cytochrome b(5) reductase and cytochrome b(5).

Abstract

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b(5) reductase and cytochrome b(5).