Active/de-active state transition of the mitochondrial complex I as revealed by specific sulfhydryl group labeling
Active/de-active state transition of the mitochondrial complex I as revealed by specific sulfhydryl group labeling
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DOI：
10.1016/s0014-5793(99)00850-9
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发表时间：
1999-07-16
期刊：
影响因子：
3.5
通讯作者：
Vinogradov, AD
中科院分区：
文献类型：
--
作者：
Gavrikova, EV;Vinogradov, AD
研究了亚线粒体颗粒和纯化的复合物I的NADH氧化酶和/或NADH-泛醌还原酶活性对N-乙基马来酰亚胺（NEM）和其他SH-试剂的敏感性。只有热失活的制剂[A. D. Vinogradov（1998）Biochim，Biophys，Acta 1364，169-185]被SH-试剂抑制，而氧化还原脉冲活化的酶对抑制剂有抗性。假一级抑制速率的pH曲线表明，对于失活依赖性的、NEM反应性巯基，约10的pK（a），用过量的NEM处理然后去除抑制剂的活化颗粒的NADH-泛醌还原酶仍然能够缓慢可逆地活化/失活转变。当活化时，经NEM处理的颗粒被失活并进一步被N-荧光素马来酰亚胺抑制。比较亚线粒体颗粒、粗品和纯化的复合物I的特异性荧光标记表明，活性/去活性状态依赖的SH-基团位于15 kDa的多肽中（最可能在复合物I的含铁-硫蛋白部分的15 kDa IP亚基中），（C）1999欧洲生物化学学会联合会。
The sensitivities of NADH oxidase and/or NADH-ubiquinone reductase activities of submitochondrial particles and purified complex I towards N-ethylmaleimide (NEM) and other SH-reagents were studied, Only thermally de-activated preparations [A.D. Vinogradov (1998) Biochim, Biophys, Acta 1364, 169-185] were inhibited by SH-reagents whereas the redox-pulsed, activated enzyme was resistant to the inhibitors. The pH profile of the pseudo-first order inhibition rate suggested a pK(a) of about 10 for the de-activation-dependent, NEM-reactive sulfhydryl group, NADH-ubiquinone reductase of activated particles treated with an excess of NEM followed by removal of the inhibitor was still capable of slow reversible active/de-active transition, When active, NEM-treated particles were de-activated and further inhibited by N-fluorescein maleimide, specific incorporation of the fluorescence label into low molecular mass polypeptide was evident, Comparison of the specific fluorescence labeling of submitochondrial particles, crude and purified complex I showed that the active/de-active state-dependent SH-group is located in a 15 kDa polypeptide (most likely in the 15 kDa IP subunit of the iron-sulfur protein-containing fraction of complex I), (C) 1999 Federation of European Biochemical Societies.