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.
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.
Mariottini
P
P
Chomyn
A
A
Doolittle
R F
RF
Attardi
G
G
D000595:Amino Acid Sequence; D000906:Antibodies; D003576:Electron Transport Complex IV / Q000032:analysis / Q000276:immunology*; D000939:Epitopes / Q000032:analysis; D006367:HeLa Cells / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D010446:Peptide Fragments / Q000276:immunology*
D000906:Antibodies; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D003576:Electron Transport Complex IV