MEMBRANE-POTENTIAL GENERATION COUPLED TO OXIDATION OF EXTERNAL NADH IN LIVER-MITOCHONDRIA

Oxidation of added NADH by rat liver mitochondria has been studied. It is found that exogenous NADH, when oxidized by rat liver mitochondria in sucrose hypotonic medium supplemented with Mg2+ and EGTA, generate s a membrane potential (Delta Psi) even in the absence of added cytoch rome c, ADP and phosphate decrease Delta Psi, the effect being reverse d by oligomycin, Rotenone and myxothiazol do not inhibit Delta Psi gen erated by oxidation of exogenous NADH. Added cytochrome c increases th e rate of the exogenous NADH oxidation and coupled Delta Psi formation . In sucrose isotonic medium, or in hypotonic medium without Mg2+, exo genous NADH fails to stimulate respiration and to form a membrane pote ntial. In the presence of Mg2+, exogenous NADH appears to be effective in Delta Psi generation in isotonic sucrose medium if mitochondria we re treated with digitonin, In isotonic KCI without Mg2+, oxidation of exogenous NADH is coupled to the Delta Psi formation and MgCl2 additio n before mitochondria prevents this effect, In hypotonic (but not in i sotonic) sucrose medium, Mg2+ makes a portion of the cytochrome c pool reducible by exogenous NADH or ascorbate, It is assumed that (i) hypo tonic treatment or digitonin causes disruption of the outer mitochondr ial membrane, and, as a consequence, desorption of the membrane-bound cytochrome c in a Mg2+-dependent fashion; (ii) incubation in isotonic KCl without Mg2+ results in swelling of mitochondrial matrix, disrupti on of the outer membrane and cytochrome c desorption whereas Mg2+ lowe rs the K+ permeability of the inner membrane and, hence, prevents swel ling; (iii) desorbed cytochrome c is reduced by added NADH via NADH-cy tochrome b(5) reductase and cytochrome b(5) or by ascorbate and is oxi dized by cytochrome oxidase, The role of desorbed cytochrome c in oxid ation of superoxide and cytoplasmic NADH as well as possible relations of these events to apoptosis are discussed, (C) 1998 Federation of Eu ropean Biochemical Societies.