Yu L, Yu C A
J Biol Chem. 1982 Feb 25;257(4):2016-21.
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.
通过在pH 8.0下用1.1% Triton X-100增溶，并在含有30 mM磷酸钾的50 mM Tris-琥珀酸缓冲液（pH 8.0）中进行磷酸钙柱色谱法，从琥珀酸-细胞色素c还原酶制备了一种纯化的活性琥珀酸-泛醌还原酶，且未对泛醇-细胞色素c还原酶造成损害。由此获得的琥珀酸-泛醌还原酶含有泛醌，并在没有外源介质的情况下催化2,6-二氯酚靛酚对琥珀酸的噻吩甲酰三氟丙酮敏感的氧化反应。添加泛醌可使活性提高约50%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明该酶含有四种多肽。在通过报道方法获得的复合物II制剂中通常观察到的高分子量多肽污染物不存在。活性琥珀酸-泛醌还原酶可以与细胞色素b-c1III复合物或复合物III重组，在没有外源泛醌的情况下形成琥珀酸-细胞色素c还原酶，或者在有泛醌和磷脂存在的情况下与解析出的泛醇-细胞色素c还原酶重组。在适当条件下，可得到所有原始的琥珀酸-细胞色素c还原酶，这表明尽管从泛醇-细胞色素c还原酶区域去除了磷脂和泛醌，但这种分离对蛋白质没有造成损害。
