| 12534287 |
Nakamaru-Ogiso E, Sakamoto K, Matsuno-Yagi A, Miyoshi H, Yagi T: The ND5 subunit was labeled by a photoaffinity analogue of fenpyroximate in bovine mitochondrial complex I. Biochemistry. 2003 Jan 28;42(3):746-54.
Fenpyroximate is a potent inhibitor of the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I). We synthesized its photoaffinity analogue [(3) H](trifluoromethyl) phenyldiazirinylfenpyroximate ([(3) H] TDF). When bovine heart submitochondrial particles (SMP) were illuminated with UV light in the presence of [(3) H] 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 [(3) H] TDF-labeled SMP 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 approximately 50 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-K (3) Fe (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. |
92(1,1,2,7) |