The ND5 subunit was labeled by a photoaffinity analogue of fenpyroximate in bovine mitochondrial complex I

被引:84
作者
Nakamaru-Ogiso, E
Sakamoto, K
Matsuno-Yagi, A
Miyoshi, H
Yagi, T [1 ]
机构
[1] Scripps Res Inst, Dept Mol & Expt Med, Div Biotechnol, La Jolla, CA 92037 USA
[2] Kyoto Univ, Grad Sch Agr, Div Appl Life Sci, Kyoto 6068502, Japan
关键词
D O I
10.1021/bi0269660
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Fenpyroximate is a potent inhibitor of the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I). We synthesized its photoaffinity analogue [H-3](trifluoromethyl)phenyldiazirinylfenpyroximate ([H-3]TDF). When bovine heart submitochondrial particles (SMP) were illuminated with UV light in the presence of [H-3]TDF, radioactivity was mostly incorporated into a 50 kDa band. There was a good correlation between radioactivity labeling of the 50 kDa band and inhibition of the NADH oxidase activity, indicating that a 50 kDa protein is responsible for the inactivation of complex I. Blue native gel electrophoresis of the [H-3]TDF-labeled SNIP revealed that the majority of radioactivity was found in complex I. Analysis of the complex I band on an SDS gel showed a major peak of radioactivity at similar to50 kDa. There are three subunits in complex I that migrate in this region: FP51K, IP49K, and ND5. Further analysis using the 2D gel electrophoresis implied that the labeled protein was the ND5 subunit. Labeling of the ND5 subunit was stimulated by NADH/NADPH but was prevented by various complex I inhibitors. Amiloride derivatives that are known to be inhibitors of Na+/H+ antiporters also diminished the labeling. In agreement with the protective effect, we observed that the amiloride derivatives inhibited NADH-ubiquinone-1 reductase activity but not NADH-K3Fe(CN)(6) reductase activity in bovine SMP. These results suggest that the ND5 subunit is involved in construction of the inhibitor- and quinone-binding site(s). Furthermore, it seems likely that the ND5 subunit may participate in H+(Na+) translocation in coupling site 1.
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页码:746 / 754
页数:9
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