Resolution and reconstitution of succinate-cytochrome c reductase: preparations and properties of high purity succinate dehydrogenase and ubiquinol-cytochrome c reductase.
An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c1 III complex). An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinae dehydrogenase fraction and the cytochrome b-c1 complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c1 complex was separated into chtochrome b-c1 III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate. The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 anbd 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low Km ferricyanide reductase activity of 85 mumol succinate oxidized per min per mg protein at 38 degrees C. Chemical composition analysis of cytochrome b-c1 III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation. When these three components were mixed in a proper ratio, a thenoyltrifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.
D003579:Cytochrome Reductases / Q000302:isolation & purification*; D008722:Methods; D009132:Muscles / Q000201:enzymology; D009245:NADH Dehydrogenase / Q000302:isolation & purification*; D010088:Oxidoreductases / Q000032:analysis*; D013384:Succinate Cytochrome c Oxidoreductase / Q000032:analysis*; D013385:Succinate Dehydrogenase / Q000302:isolation & purification*; D014451:Ubiquinone / Q000302:isolation & purification*
D014451:Ubiquinone; D010088:Oxidoreductases; D013384:Succinate Cytochrome c Oxidoreductase; D013385:Succinate Dehydrogenase; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase