Restoration of deoxycholate-disrupted membrane oxidases of Micrococcus lysodeikticus.
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.
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D001647:Bile Acids and Salts / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002850:Chromatography, Gel; D003124:Colorimetry; D003470:Culture Media; D003580:Cytochromes / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004579:Electron Transport; D004789:Enzyme Activation; D008274:Magnesium / Q000494:pharmacology*; D008278:Magnesium Sulfate; D008293:Malates; D008837:Micrococcus / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D008854:Microscopy, Electron; D009243:NAD; D010088:Oxidoreductases / Q000378:metabolism*; D010101:Oxygen Consumption; D010772:Phosphotungstic Acid; D011048:Polarography; D013053:Spectrophotometry; D013194:Staining and Labeling; D013447:Sulfites / Q000494:pharmacology; D013997:Time Factors
D001647:Bile Acids and Salts; D003470:Culture Media; D003580:Cytochromes; D008293:Malates; D013447:Sulfites; D009243:NAD; D010772:Phosphotungstic Acid; D008278:Magnesium Sulfate; D004492:Edetic Acid; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D008274:Magnesium; D002118:Calcium