Isolation and properties of the outer membrane of plant mitochondria.

The outer membrane of turnip (Brassica rapa L.) mitochondria was isolated by incubating the mitochondria with a dilute digitonin solution and differential centrifuging. The outer membrane fraction was not contaminated by inner membrane enzymes and lacked an NADPH-cytochrome c reductase. However it possessed very active NADH-cytochrome c, dichloroindophenol and ferricyanide reductases which were insensitive to antimycin A, Amytal and low (less than 10 μm) concentrations of Dicumarol. p-Chloromercuribenzoate (ClHgBzO^?) and high concentrations (greater than 10 μm) of Dicumarol inhibited the reductases, ClHgBzO^? almost completely. Preincubation of the outer membrane with NADH protected it from ClHgBzO^? inhibition. An acid phosphatase and an NADPH-ferricyanide reductase were also detected, but the latter was only loosely bound to the membrane. The NADH dehydrogenase of the outer membrane was insensitive to ethylene glycol-bis(β-aminoethyl ether)N,N′-tetraacetate (1 mm) and was not stimulated by CaCl₂ (0.5 mm), thus differing from the external NADH oxidase of the inner membrane (Coleman, J. O. D., and Palmer, J. M. (1971) FEBS Lett., 17, 203–208). Respiratory-linked oxidation of exogenous NADH by intact mitochondria showed a similar pattern of inhibition by ClHgBzO^? as did the outer membrane, but was inhibited strongly by low concentrations of Dicumarol (5 μm inhibited by 70%).