Restoration of deoxycholate-disrupted membrane oxidases of Micrococcus lysodeikticus.

Abstract

Membrane-associated l-malate and reduced nicotinamide adenine dinucleotide (NADH) oxidase complexes of Micrococcus lysodeikticus were inactivated with deoxycholate. Reactivation of NADH oxidase by addition of Mg(2+) occurred in these detergent-membrane mixtures, but reactivation of l-malate oxidase did not occur in the presence of deoxycholate. Removal of detergent by gel filtration allowed Mg(2+)-dependent restoration of both l-malate and NADH oxidases. Maximal NADH and l-malate oxidase restoration required 10 min and 40 min, respectively, at 30 mm MgSO(4). Maximal restoration of both oxidases required at least 12 mm MgSO(4) in an incubation period of 1 hr. Reduced-minus-oxidized difference spectra of Mg(2+)-restored membrane oxidases showed participation of cytochromes b, c, and a when either l-malate or NADH served as reductant; addition of dithionite did not increase the alpha- and beta-region absorbancy maxima of these hemoproteins when restored membranes were first reduced with the physiological substrates l-malate or NADH. Not all divalent cations tested were equally effective for reactivation of both oxidases. l-Malate oxidase was restored by both Mn(2+) and Ca(2+). NADH oxidase was not activated by Mn(2+) and only slightly stimulated by Ca(2+). Separation of deoxycholate-disrupted membranes (detergent removed) into soluble and particulate fractions showed that both fractions were required for Mg(2+)-dependent oxidase activities. Electron micrographs indicated conditions of detergent treatment did not destroy the vesicular nature of protoplast ghost membranes.