A preparation containing the Mr 13,400 protein (subunit VI), phospholipid, and ubiquinone was isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea solubilization, calcium phosphate-cellulose column chromatography at different pHs, acetone precipitation, and decanoyl-N-methylglucamide-sodium cholate extraction. The protein in this preparation corresponds to subunit VI of ubiquinol-cytochrome c reductase resolved in the sodium dodecyl sulfate-polyacrylamidce gel electrophoresis system of Schägger et al. (1987, FEBS Lett. 21, 161-168) and has the same amino acid sequence as that of the Mr 13,400 protein reported by Wakabayashi et al. (1985, J. Biol. Chem. 260, 337-343). The phospholipid and ubiquinone present in the preparation copurify with but are not intrinsic components of, the Mr 13,400 protein. This preparation has a potency and behavior identical to that of a free phospholipid preparation in restoring activity to delipidated ubiquinol-cytochrome c reductase. Antibodies against Mr 13,400 react only with Mr 13,400 protein and complexes which contain it. They do not inhibit intact, lipid-sufficient ubiquinol-cytochrome c reductase. However, when delipidated ubiquinol-cytochrome c reductase is incubated with antibodies prior to reconstitution with phospholipid, a 55% decrease in the restoration activity is observed, indicating that the catalytic site-related epitopes of the Mr 13,400 protein are buried in the phospholipid environment. Antibodies against Mr 13,400 cause an increase of apparent Km for ubiquinol-2 in ubiquinol-cytochrome c reductase. When mitoplasts or submitochondrial particles are exposed to a horseradish peroxidase conjugate of the Fab' fragment of anti-Mr 13,400 antibodies, peroxidase activity is found mainly in the submitochondrial particles preparation; little activity is detected in mitoplasts. This suggests that the Mr 13,400 protein is extruded toward the matrix side of the membrane.

译文

通过涉及Triton X-100和尿素增溶,磷酸钙-纤维素柱色谱在不同ph下,丙酮沉淀,从牛心线粒体泛醇-细胞色素c还原酶中分离出含有Mr 13,400蛋白 (亚基VI),磷脂和泛醌的制剂,和癸酰基-N-甲基葡糖酰胺-胆酸钠提取。该制剂中的蛋白质对应于在sch ä gger等人的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳系统中解析的泛醇-细胞色素c还原酶的亚基VI (1987,FEBS Lett. 21,161-168),并且具有与Wakabayashi等人报道的Mr 13,400蛋白相同的氨基酸序列 (1985,J. Biol. Chem. 260,337-343)。制剂中存在的磷脂和泛醌与Mr 13,400蛋白共纯化但不是固有成分。该制剂在恢复脱脂的泛醇-细胞色素c还原酶的活性方面具有与游离磷脂制剂相同的效力和行为。抗Mr 13,400的抗体仅与Mr 13,400蛋白和含有它的复合物反应。它们不抑制完整的,脂质充足的泛醇-细胞色素c还原酶。然而,当脱脂的泛醇-细胞色素c还原酶在用磷脂重建之前与抗体孵育时,观察到恢复活性的55% 降低,表明Mr 13,400蛋白的催化位点相关表位被掩埋在磷脂环境中。抗Mr 13,400的抗体导致泛醇-细胞色素c还原酶中ubiquinol-2的表观Km增加。当mitoposal或线粒体颗粒暴露于抗Mr 13,400抗体的Fab' 片段的辣根过氧化物酶缀合物时,过氧化物酶活性主要存在于线粒体颗粒制剂中; 在mitoposal中检测到的活性很少。这表明Mr 13,400蛋白向膜的基质侧挤出。

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