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 m m MgSO 4 . Maximal restoration of both oxidases required at least 12 m m 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 α- and β-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.