Reconstitution of Micrococcus lysodeikticus reduced nicotinamide adenine dinucleotide and L-malate dehydrogenases with dehydrogenase-depleted membrane residues: a basis for restoration of oxidase activities.

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.