Synthesis of functional probes toward elucidation of the function of respiratory enzymes of Caenorhabditis elegans.

为了确定甲氰菊酯的结合亚基，它是牛心线粒体复合体I的一种非常有效的抑制剂，我们合成了~3H标记的甲氰菊酯([~3H](三氟甲基)-苯基二氮丙基甲酯，TDF)。TDF对牛心线粒体复合体I有很强的抑制作用，几乎等同于苯丙酮肟。在与美国斯克里普斯研究所的Takao Yagi博士合作对牛心复合体I进行光亲和研究的基础上，TDF的结合位点被证明是存在于酶的膜臂中的ND5亚基。由于抗ND5亚基C末端的抗体只能识别ND5亚基，因此我们用2D-PAGE(十二烷基硫酸钠-PAGE)对ND5进行鉴定。我们的结果是首次将位于膜臂上的亚基确定为抑制物结合亚基。我们的研究有力地表明，位于膜臂上的亚基(S)可能参与了络合物I的质子泵活性。

To identify the binding subunit of fenpyroximate, a very potent inhibitor of bovine heart mitochondrial complex I, we synthesized ^3H-labeled photolabile fenpyroximate ([^3H](trifluoromethyl)-phenyldiazirinyl fenpyroximate, TDF). TDF appeared to elicit very potent inhibition of bovine heart mitochondrial complex I being nearly equivalent to that of fenpyroximate.On the basis of photoaffinity studies with bovine heart complex I in collaboration with Dr.Takao Yagi (The Scripps Research Institute, USA), the binding site of TDF was shown to be subunit ND5 which is thought to exist in membrane arm of the enzyme. Since antibody to the C-terminal region of the ND5 subunit only weakly recognized the subunit, we identified the ND5 by 2D-electrophoresis (SDS-PAGE). Our result is the first study wherein the subunit located in membrane arm was identified as the inhibitor binding subunit. Our study strongly suggests that subunit(s) located in membrane arm may be involved in the proton-pumping activity of complex I.