Triton X-100 as a specific inhibitor of the mammalian NADH-ubiquinone oxidoreductase (Complex I).
Triton X-100 as a specific inhibitor of the mammalian NADH-ubiquinone oxidoreductase (Complex I).
复制标题
Triton X-100 作为哺乳动物 NADH-泛醌氧化还原酶（复合物 I）的特异性抑制剂。
DOI：
10.1016/s0005-2728(98)00156-x
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Vinogradov,AD
中科院分区：
文献类型：
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作者：
Ushakova,AV;Grivennikova,VG;Ohnishi,T;Vinogradov,AD
Triton X-100 抑制牛心脏软骨下颗粒 (SMP) 的 NADH 氧化酶和鱼藤酮敏感的 NADH-Q1 还原酶活性，表观 Kiof 1×10−5M（pH 8.0，25°C）。紧密耦合 SMP 中的 NADH-六氨合钌还原酶、琥珀酸氧化酶以及以琥珀酸为底物的呼吸控制比在抑制剂浓度低于 0.15 mM 时不受影响。琥珀酸支持的有氧反向电子转移对抑制剂 (Ki=5×10−5M) 的敏感性低于 NADH 氧化酶。与鱼藤酮类似，当将核苷酸添加到紧密耦合的 SMP 有氧氧化琥珀酸中时，有限浓度的 Triton X-100 会增加 NAD+ 还原的稳态水平。与鱼藤酮类似，Triton X-100 部分保护复合物 I 免受热诱导失活，并部分激活热失活酶。在 Triton X-100 存在的情况下，鱼藤酮对 NADH 氧化酶的抑制率急剧下降，这表明这两种抑制剂之间存在对共同特异性结合位点的竞争。与鱼藤酮相反，Triton X-100 的抑制作用在反应混合物稀释后立即逆转。 SMP 的 NADH-Q1 还原酶活性被添加的 Q1 非竞争性抑制，而对于分离的复合物 I，可以看到 Q1 和抑制剂之间的简单竞争。获得的结果表明，Triton X-100 是复合物 I 泛醌还原的特异性抑制剂，并且与我们之前的发现一致，即不同的反应途径在哺乳动物呼吸链的这一段的正向和反向电子转移中起作用。
Triton X-100 inhibits the NADH oxidase and rotenone-sensitive NADH-Q1reductase activities of bovine heart submitochondrial particles (SMP) with an apparent Kiof 1×10−5M (pH 8.0, 25°C). The NADH-hexammineruthenium reductase, succinate oxidase, and the respiratory control ratio with succinate as the substrate in tightly coupled SMP are not affected at the inhibitor concentrations below 0.15 mM. The succinate-supported aerobic reverse electron transfer is less sensitive to the inhibitor (Ki=5×10−5M) than NADH oxidase. Similar to rotenone, limited concentrations of Triton X-100 increase the steady-state level of NAD+reduction when the nucleotide is added to tightly coupled SMP oxidizing succinate aerobically. Also similar to rotenone, Triton X-100 partially protects Complex I against the thermally induced deactivation and partially activates the thermally deactivated enzyme. The rate of the NADH oxidase inhibition by rotenone is drastically decreased in the presence of Triton X-100 which indicates a competition between these two inhibitors for a common specific binding site. In contrast to rotenone, the inhibitory effect of Triton X-100 is instantly reversed upon dilution of the reaction mixture. The NADH-Q1reductase activity of SMP is inhibited non-competitively by added Q1whereas a simple competition between Q1and the inhibitor is seen for isolated Complex I. The results obtained show that Triton X-100 is a specific inhibitor of the ubiquinone reduction by Complex I and are in accord with our previous findings which suggest that different reaction pathways operate in the forward and reverse electron transfer at this segment of the mammalian respiratory chain.