Modulation of oxidative phosphorylation of human kidney 293 cells by transfection with the internal rotenone-insensitive NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae

被引:50
作者
Seo, BB [1 ]
Matsuno-Yagi, A [1 ]
Yagi, T [1 ]
机构
[1] Scripps Res Inst, Dept Expt & Mol Biol, Div Biochem, La Jolla, CA 92037 USA
来源
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS | 1999年 / 1412卷 / 01期
关键词
NADH dehydrogenase; mammalian cell; mitochondrial disease; oxidative phosphorylation; gene therapy;
D O I
10.1016/S0005-2728(99)00051-1
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
In contrast to the mitochondrial proton-translocating NADH-quinone oxidoreductase complex I!, which consists of at least 43 different subunits, the internal rotenone-insensitive NADH-quinone oxidoreductase (Ndil) of Saccharomyces cerevisiae is a single polypeptide enzyme. The NDI1 gene was stably transfected into the human embryonal kidney 293 (HEK 293) cells. The transfected NDI1 gene was then transcribed and translated in the HEK 293 cells to produce the functional enzyme. The immunochemical and immunofluorescence analyses indicated that the expressed Ndil polypeptide was located to the inner mitochondrial membranes. The expression of Ndil did not alter the content of existing complex I in the HEI 293 mitochondria, suggesting that the expressed Ndil enzyme does not displace the endogenous complex I. The NADH oxidase activity of the NDI1-transfected HEK 293 cells was not affected by rotenone but was inhibited by flavone. The ADP/O ratios coupled to NADH oxidation were lowered from 2.4 to 1.8 by NDI1-transfection while the ADP/O ratios coupled to succinate oxidation (1.6) were not changed. The NDI1-transfected HEK 293 cells were able to grow in media containing a complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. The potential usefulness of incorporating the Ndil protein into mitochondria of human cells is discussed. (C) 1999 Elsevier Science B.V. All rights reserved.
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页码:56 / 65
页数:10
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