Immunochemical study of subunit VI (Mr 13,400) of mitochondrial ubiquinol-cytochrome c reductase.
A preparation containing the Mr 13,400 protein (subunit VI), phospholipid, and ubiquinone was isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea solubilization, calcium phosphate-cellulose column chromatography at different pHs, acetone precipitation, and decanoyl-N-methylglucamide-sodium cholate extraction. The protein in this preparation corresponds to subunit VI of ubiquinol-cytochrome c reductase resolved in the sodium dodecyl sulfate-polyacrylamidce gel electrophoresis system of Schägger et al. (1987, FEBS Lett. 21, 161-168) and has the same amino acid sequence as that of the Mr 13,400 protein reported by Wakabayashi et al. (1985, J. Biol. Chem. 260, 337-343). The phospholipid and ubiquinone present in the preparation copurify with but are not intrinsic components of, the Mr 13,400 protein. This preparation has a potency and behavior identical to that of a free phospholipid preparation in restoring activity to delipidated ubiquinol-cytochrome c reductase. Antibodies against Mr 13,400 react only with Mr 13,400 protein and complexes which contain it. They do not inhibit intact, lipid-sufficient ubiquinol-cytochrome c reductase. However, when delipidated ubiquinol-cytochrome c reductase is incubated with antibodies prior to reconstitution with phospholipid, a 55% decrease in the restoration activity is observed, indicating that the catalytic site-related epitopes of the Mr 13,400 protein are buried in the phospholipid environment. Antibodies against Mr 13,400 cause an increase of apparent Km for ubiquinol-2 in ubiquinol-cytochrome c reductase. When mitoplasts or submitochondrial particles are exposed to a horseradish peroxidase conjugate of the Fab' fragment of anti-Mr 13,400 antibodies, peroxidase activity is found mainly in the submitochondrial particles preparation; little activity is detected in mitoplasts. This suggests that the Mr 13,400 protein is extruded toward the matrix side of the membrane.
Usui
S
S
Department of Biochemistry, Oklahoma State University, Stillwater 74078.
Yu
L
L
Harmon
J
J
Yu
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D000918:Antibody Specificity; D002417:Cattle; D014450:Electron Transport Complex III / Q000032:analysis* / Q000037:antagonists & inhibitors; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000201:enzymology; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017830:Octoxynol; D010743:Phospholipids / Q000302:isolation & purification; D011092:Polyethylene Glycols; D011506:Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D012995:Solubility; D013384:Succinate Cytochrome c Oxidoreductase / Q000037:antagonists & inhibitors; D014451:Ubiquinone / Q000302:isolation & purification; D014508:Urea
D000906:Antibodies; D010743:Phospholipids; D011506:Proteins; D014451:Ubiquinone; D011092:Polyethylene Glycols; D014508:Urea; D017830:Octoxynol; D013384:Succinate Cytochrome c Oxidoreductase; D014450:Electron Transport Complex III