Quantitative resolution of succinate-cytochrome c reductase into succinate-ubiquinone and ubiquinol-cytochrome c reductases.
A purified, active succinate-ubiquinone reductase was prepared from succinate-cytochrome c reductase without damage to ubiquinol-cytochrome c reductase by 1.1% Triton X-100 solubilization at pH 8.0, and calcium phosphate column chromatography in 50 mM Tris-succinate buffer, pH 8.0, containing 30 mM potassium phosphate. Succinate-ubiquinone reductase thus obtained contains ubiquinone and catalyzes thenoyltrifluoroacetone-sensitive oxidation of succinate by 2,6-dichlorophenolindophenol in the absence of exogenous mediator. Addition of ubiquinone enhanced the activity about 50%. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme contains four polypeptides. The high molecular weight polypeptide contaminants usually observed in the Complex II preparation obtained by the reported method were absent. The active succinate-ubiquinone reductase can reconstitute with the cytochrome b-c1III complex, or Complex III to form succinate-cytochrome c reductase in the absence of exogenous ubiquinone or with the resolved ubiquinol-cytochrome c reductase in the presence of ubiquinone and phospholipids. Under the proper conditions, all the original succinate-cytochrome c reductase was obtained, indicating that the resolution caused no damage to the protein, despite the removal of phospholipids and ubiquinone from the ubiquinol-cytochrome c reductase region.
D000818:Animals; D002417:Cattle; D042963:Electron Transport Complex II; D014450:Electron Transport Complex III; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D011808:Quinone Reductases / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D013367:Submitochondrial Particles / Q000201:enzymology*; D013384:Succinate Cytochrome c Oxidoreductase / Q000302:isolation & purification / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*
D009097:Multienzyme Complexes; D010088:Oxidoreductases; D013384:Succinate Cytochrome c Oxidoreductase; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III