Cytochrome c oxidase biosynthesis and assembly in Candida utilis yeast cells : Subunit structure and molecular weight determination

Abstract

Cytochrome c oxidase was purified from mitochondria of Candida utilis yeast cells. The purification procedure involved the hypotonic incubation of mitochondria followed by washes with increasing concentrations of KCl. The membrane fragments derived from this procedure were subjected to ammonium sulfate fractionation in the presence of 2% cholate. The purified active enzyme contained 8.5–9.2 nmol heme a per mg protein and was free of other types of hemoproteins. Upon Sephadex G200 gel filtration in the presence of cholate, an apparent molecular weight of 200,000 was estimated. A single band was observed for the active enzyme upon DEAE-cellulose chromatography, sucrose density gradient centrifugation, and Sephadex G200 gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate resolved the enzyme into six polypeptide bands with apparent molecular weights of 49,000, 32,000, 28,000, 20,000, 13,500, and 8,000, respectively. The six components were also resolved by gel filtration on Sephadex G200, equilibrated with 0.1% sodium dodecyl sulfate, giving apparent molecular weights of 46,000, 35,000, 23,000, 19,000, 12,500, and 7,800.