从亚线粒体颗粒中分离F1因子的α亚基，并从分离的α亚基和结合在线粒体内膜上的β亚基中重建活性ATP酶。
Isolation of alpha-subunits of factor F1 from submitochondrial particles and the reconstitution of active ATPase from isolated alpha-subunits and beta-subunits bound to the mitochondrial membrane.
作者信息
Kozlov I A, Milgrom Y M, Tsybovski I S
出版信息
Biochem J. 1980 Nov 15;192(2):483-8. doi: 10.1042/bj1920483.
Abstract
The alpha-subunits of factor-F1 ATPase are removed by extraction of submitochondrial particles with 1.75 M-LiCl, with the consequent loss of ATPase activity. ATPase activity is reconstituted by incubation of LiCl-extracted particles with purified alpha-subunits, and the reconstituted ATPase activity is oligomycin-sensitive. Reconstitution is enhanced by maintenance of the alpha-subunits in reduced form by dithiothreitol or NaBH4 and by modification of the alpha-subunits by p-chloromercuribenzoate, iodoacetic acid or N-ethylmaleimide. Experiments with the mixed anhydride of ATP and mesitylene-carboxylic acid, which was previously shown to interact with the F1 active site, localized on the beta-subunits, indicate that the active site of ATPase is shielded by the alpha-subunits.
摘要
用1.75M-LiCl提取亚线粒体颗粒可去除F1-ATP酶的α亚基，随之ATP酶活性丧失。通过将LiCl提取的颗粒与纯化的α亚基一起温育可重建ATP酶活性，且重建的ATP酶活性对寡霉素敏感。用二硫苏糖醇或硼氢化钠将α亚基维持在还原形式，以及用对氯汞苯甲酸、碘乙酸或N-乙基马来酰亚胺修饰α亚基，均可增强重建效果。先前已证明ATP与均三甲苯羧酸的混合酸酐可与位于β亚基上的F1活性位点相互作用，对其进行的实验表明，ATP酶的活性位点被α亚基所屏蔽。
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