Purification and polypeptide characterization of complex III from yeast mitochondria.
Complex III was isolated and purified from bakers' yeast by ammonium sulfate fractionation and column chromatography on Ultrogel AcA 34. The purified complex contained 7.03 nmol/mg of protein and 4.24 nmol/mg of protein of cytochromes b and c1, respectively. The specific activity of the complex was 17.1 mumol/min/mg of protein, using the decyl analog of coenzyme Q as substrate. Electrophoresis of the purified complex revealed the presence of seven polypeptides with molecular weights ranging from 15,500 to 50,000. Polypeptides having molecular weights lower than 15,000 were not observed, except when the complex was dissociated in the absence of proteolytic inhibitors, suggesting that these low molecular weight species arise as a result of proteolytic digestion of the complex. The isoelectric points of the subunits of complex III and their stoichiometry wee determined. Trypsin and chymotrypsin digestion of the oxidized and reduced forms of the isolated complex suggested that the two high molecular weight core proteins are embedded within the complex and hence are inaccessible to the exogenous proteases, while cytochromes b and c1, the iron-sulfur protein, and the 17,500-dalton subunit are substantially exposed to the surface of the complex. The iron-sulfur protein appears to undergo a conformational change upon reduction of the complex, rendering it less susceptible to trypsin digestion. The core proteins and the iron-sulfur protein were purified, and antibodies against these proteins were raised. Immunoinhibition studies with these antibodies also indicated that the antigenic sites of the core proteins were embedded in the complex.
D000936:Antigen-Antibody Complex; D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism; D003575:Cytochromes c1 / Q000378:metabolism; D014450:Electron Transport Complex III; D007106:Immune Sera; D005779:Immunodiffusion; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D011808:Quinone Reductases / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000936:Antigen-Antibody Complex; D003573:Cytochrome b Group; D003580:Cytochromes; D007106:Immune Sera; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D003575:Cytochromes c1; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III