Mariottini, Paolo and Chomyn, Anne and Doolittle, Russell F. and Attardi, Giuseppe (1986) Antibodies against the COOH-terminal undecapeptide of subunit II, but not those against the NH2-terminal decapeptide, immunoprecipitate the whole human cytochrome c oxidase complex. Journal of Biological Chemistry, 261 (7). pp. 3355-3362. ISSN 0021-9258 http://resolver.caltech.edu/CaltechAUTHORS:MARjbc86
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Antibodies against synthetic peptides derived from the DNA sequence of human cytochrome c oxidase subunit II (COII) have been tested for their capacity to immunoprecipitate the whole enzyme complex. Antibodies against the COOH-terminal undecapeptide of COII (anti-COII-C), when incubated with a Triton X-100 mitochondrial lysate from HeLa cells pulse-labeled with [35S]methionine under conditions selective for mitochondrial protein synthesis and chased for 18 h in unlabeled medium, precipitated the pulse-labeled three largest subunits (mitochondrially synthesized) of cytochrome c oxidase in proportions close to equimolarity. Antibodies against the NH2-terminal decapeptide of COII (anti-COII-N), although equally reactive as the anti-COII-C antibodies with the sodium dodecyl sulfate-solubilized COII, did not precipitate any of the three labeled subunits from the Triton X-100 mitochondrial lysate. In other experiments, all the 13 subunits which have been identified in the mammalian cytochrome c oxidase were immunoprecipitated from a Triton X-100 mitochondrial lysate of cells long-term labeled with [35S]methionine by anti-COII-C antibodies, but not by anti-COII-N antibodies. By contrast, in immunoblots of total mitochondrial proteins dissociated with sodium dodecyl sulfate, the anti-COII-C antibodies reacted specifically only with COII. These results strongly suggest that, in the native cytochrome c oxidase complex, the epitope recognized by the anti-COII-C antibodies is in the COII subunit and that, therefore, in such complex, the COOH-terminal peptide of COII is exposed to antibodies, whereas the NH2-terminal peptide is not accessible.
|Additional Information:||© 1986 American Society of Biological Chemists. Received for publication, July 15, 1985. We are very grateful to Dr. Edwin Ching for a gift of bovine and human cytochrome c oxidase. The excellent assistance of Doris Finch and Arger Drew is gratefully acknowledged. This work was supported by National Institutes of Health Grant GM-11726 (to G.A.) and National Science Foundation Grant PCM-8118172 (to R.F.D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.|
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|Deposited By:||Tony Diaz|
|Deposited On:||11 Dec 2008 23:58|
|Last Modified:||26 Dec 2012 10:30|
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