Chemical modification of mitochondria: I. Specific labeling by 2,4-dinitro-5-(bromo-[2-14C]acetoxyethoxy) phenol |
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Authors: | Jui H. Wang Osamu Yamauchi Shu-I Tu Kuan Wang David R. Saunders Les Copeland Eva Copeland |
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Affiliation: | Bioenergetics Laboratory, Acheson Hall, State University of New York, Buffalo, New York 14214 U.S.A. |
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Abstract: | The uncoupler dinitrobromoacetoxyethoxyphenol (DNBP) has been synthesized and found to label rapidly and specifically a small number of cysteine residues in rat liver mitochondria. The labeling reaction was essentially complete in a few minutes. Only eight of the mitochondrial polypeptide bands, of MW 97, 58, 52, 43, 30, 26, 22, 13 × 103, respectively, as separated by SDS gel electrophoresis, were found to carry the radioactive label. In each case, the label which survived acid hydrolysis was covalently bound to cysteine residues through alkylation reaction. Under the present experimental conditions, only 0.45 mole of the label was covalently bound to mitochondrial protein per mole of cytochrome aa3 and only 1 out of about 650 sulfhydryl groups was so labeled. Consideration of the specificity of the labeling and the observed time-dependence of DNBP-stimulated respiration suggests that the labeled protein molecules are either at or very close to the mitochondrial coupling sites and probably play an important role in the primary energy transduction process. |
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