Ubiquinone binding domains in bovine heart mitochondrial cytochrome b.
Cytochrome b was identified as one of the ubiquinone-binding proteins in bovine heart mitochondrial ubiquinol-cytochrome c reductase by photoaffinity labeling using 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]-octyl)-1,4-benzoquinone ([3H]azido-Q). The [3H]azido-Q-labeled cytochrome b protein was purified to homogeneity from the azido-Q-labeled ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea treatment, calcium phosphate column chromatography, acetone precipitation, decanoyl-N-methylglucamide-cholate extraction, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified cytochrome b protein containing 0.5 mol of azido-Q/mol of protein was subjected to reductive carboxymethylation and succinylation prior to digestion by chymotrypsin. Two azido-Q-linked peptides with retention times of 47.1 and 49.0 min were obtained by high performance liquid chromatographic separation. Partial amino-terminal amino acid sequences of these two peptides were determined to be GATVI- and ALVADL-, indicating that these two chymotryptic peptides are from amino residues 142-155 and 326-336. Monospecific polyclonal antibodies against two synthetic ubiquinone-binding peptides, NH2-G-A-T-V-I-T-N-L-L-S-COOH (P-47) and NH2-W-A-L-V-A-D-L-L-T-L-T-W-I-COOH (P-49), were generated in rabbits and purified. Western blotting and enzyme-linked immunosorbent assays showed that the purified antibodies against P-47 reacted with cytochrome b-containing reductases and purified cytochrome b protein. Antibodies against P-47 inhibited activities of succinate-cytochrome c and ubiquinol-cytochrome c reductases only when they were incubated with phospholipid-depleted reductases prior to the replenishment with phospholipid. No inhibition was observed with incubation with phospholipid-containing reductases, indicating that this peptide involved in ubiquinone binding is buried in a phospholipid environment.
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Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078-0454.
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism*; D014450:Electron Transport Complex III / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism*
D000906:Antibodies; D003573:Cytochrome b Group; D009842:Oligopeptides; D010446:Peptide Fragments; D014451:Ubiquinone; D002918:Chymotrypsin; D014450:Electron Transport Complex III