Reconstitution of Micrococcus lysodeikticus Reduced Nicotinamide Adenine Dinucleotide and l -Malate Dehydrogenases with Dehydrogenase-Depleted Membrane Residues: a Basis for Restoration of Oxidase Actitivies

Abstract

Deoxycholate disruption of Micrococcus lysodeikticus protoplast membranes resulted in solubilization of both l -malate and reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase enzymes (substrate: 2,6-dichlorophenolindophenol oxidoreductases). Insoluble residues contained cytochromes of the b, c , and a type. Solubilized dehydrogenases were reconstituted with insoluble residues by treatment of disrupted membranes with magnesium ions. Most of the solubilized l -malate and NADH dehydrogenase activities were precipitated by magnesium ions independent of enzyme reconstitution with insoluble residues. Reconstituted dehydrogenases explained the mechanism for restoration of disrupted l -malate and NADH oxidase activities (4). Black light irradiation inhibited oxidase activities of both native and reconstituted membranes. These irradiated membrane oxidases were partially restored by exogenous napthoquinones [K 2(20) and K 2(50) ] but not by CoQ (6) . Reconstitution experiments showed that native membrane napthoquinone was retained in the insoluble residues of deoxycholate-disrupted membranes.