Cytochrome c1 of bakers' yeast. I. Isolation and properties.
Cytochrome c1 has been purified from mitochondria of the yeast Saccharomyces cerevisiae. The procedure involves solubilization withcholate, ammonium sulfate fractionation, disruption of the dytochrome b-c1 complex with mercaptoethanol and detergents, and chromatography on DEAE-cellulose. The final product is psectrally pure, contains up to 62 nmol of covalently bound heme per mg of protein and does not react with oxygen or carbon monoxide. Sodium dodecyl sulfate disaggregates the purified cytochrome into a single 31,000 dalton subunit carrying the covalently attached heme group. Many cytochrome c1 preparations contain in addition an 18,500 dalton polypeptide which is devoid of covalently bound heme. Since this polypeptide can be removed from the heme-carrying polypeptide by relatively mild procedures, it is probably not an essential subunit of cytochrome c1. Cytochrome c1 is extremely sensitive to proteolysis. If it si purified in the absence of protease inhibitors, a family of heme polypeptides with molecular weights of 29,000, 27,000, and 25,000 daltons is obtained. In the presence of the protease inhibitor phenylmethylsulfonylfluoride the purification yields predominantly a 31,000 dalton heme protein with only little contamination by a 29,000 dalton degradation product. In order to show that only the 31,000 dalton heme-polypeptide is the native species, yeast cells were labeled with the heme-precursor delta-amino[3H]levulinic acid, converted to protoplasts and directly lysed with dodecyl sulfate in the presence of protease inhibitors. Subsequent electrophoresis of the lysate in the presence of dodecyl sulfate reveals the covalently bound heme of cytochrome c1 as a single symmetrical peak at 31,000 daltons.
D000596:Amino Acids / Q000032:analysis; D003574:Cytochrome c Group / Q000031:analogs & derivatives*; D003575:Cytochromes c1 / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D000596:Amino Acids; D003574:Cytochrome c Group; D046911:Macromolecular Substances; D003575:Cytochromes c1