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首页> 外文期刊>Biochemistry >The ND1 Subunit Constructs the Inhibitor Binding Domain in Bovine Heart Mitochondrial Complex I
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The ND1 Subunit Constructs the Inhibitor Binding Domain in Bovine Heart Mitochondrial Complex I

机译：ND1亚基在牛心脏线粒体复合体I中构建抑制剂结合结构域

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The inhibitor binding domain in bovine complex I is believed to be constructed by multisubunits, but it remains to be learned how the binding positions of chemically diverse inhibitors relate to each other. To get insight into the inhibitor binding domain in complex I, we synthesized a photoreactive acetogenin [[~(125)I](trifluoromethyl)phenyldiazirinylacetogenin, [~(125)I]TDA], in which an aryldiazirine group serves as both a photoreactive group and a substitute for the gamma-lactone ring that is a common toxophore of numerous natural acetogenins, and carried out photoaffinity labeling to identify the labeled subunit using bovine heart submitochondrial particles (SMP). When SMP were UV-irradiated in the presence of [~(125)I]TDA, radioactivity was predominantly incorporated into an ~30 kDa band on a SDS gel. Blue native gel electrophoresis of the [~(125)I]TDA-labeled SMP revealed that the majority of radioactivity was observed in complex I. Analysis of complex I on a SDS gel showed a predominant peak of radioactivity at ~30 kDa. Immnoprecipitation of the [~(125)I]TDA-labeled complex I with anti-bovine ND1 antibody indicated that the labeled protein is the ND1 subunit. A variety of complex I inhibitors such as piericidin A and rotenone efficiently suppressed the specific binding of [~(125)I]TDA to ND1, indicating that they share a common binding domain. However, the suppression efficiency of DELTA lac-acetogenin, a new type of complex I inhibitor synthesized in our laboratory, was much lower than that of the traditional inhibitors. Our results unequivocally reveal that the ND1 subunit constructs the inhibitor binding domain, though the contribution of this subunit has been challenged. Further, the present study corroborates our previous proposition that the inhibition site of DELTA lac-acetogenins differs from that of traditional inhibitors.
机译：牛复合物I中的抑制剂结合结构域被认为是由多亚基构建的，但是化学上不同的抑制剂的结合位置如何相互关联还有待了解。为了深入了解复合物I中的抑制剂结合域，我们合成了一种光反应性产乙酸原素[[〜（125）I]（三氟甲基）苯基二叠氮基产乙酸素，[〜（125）I] TDA]，其中芳基二叠氮基同时具有光反应性γ-内酯环的替代物，γ-内酯环是许多天然产乙酸原素的常见毒素，并进行了光亲和标记以使用牛心脏线粒体颗粒（SMP）识别标记的亚基。当在[〜（125）I] TDA存在下对SMP进行紫外线照射时，放射性主要掺入了SDS凝胶上的〜30 kDa谱带中。 [〜（125）I] TDA标记的SMP的蓝色天然凝胶电泳显示，在复合物I中观察到了大多数放射性。在SDS凝胶上对复合物I进行的分析显示，在〜30 kDa处有一个主要的放射性峰。 [〜（125）I] TDA标记的复合物I与抗牛ND1抗体的免疫沉淀表明标记的蛋白是ND1亚基。各种复杂的I抑制剂（例如Piericidin A和鱼藤酮）可有效抑制[〜（125）I] TDA与ND1的特异性结合，表明它们共享一个共同的结合域。但是，在我们实验室合成的新型复合物I抑制剂DELTA lac-acetogenin的抑制效率远低于传统抑制剂。我们的结果清楚地表明，尽管该亚基的贡献受到了挑战，但ND1亚基构建了抑制剂结合域。此外，本研究证实了我们先前的命题，即DELTA lac-acetogenins的抑制位点与传统抑制剂不同。

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