Cytochrome c oxidase from the slime mold Dictyostelium discoideum: Purification and characterization

Cytochrome c oxidase was purified from growing cells of the slime mold Dictyostelium discoideum by a procedure based on hydrophobic and affinity chromatography. A highly pure (13.4-15 nmol of heme a/mg of protein) and active (turnover number = 280-330 s-1, when assayed polarographically with the slime mold cytochrome c) enzyme preparation was obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under conditions where the 12 polypeptide components of the bovine enzyme are resolved, shows that the amoeba oxidase consists of six subunits with molecular masses of 55, 29.5,19,13,11, and 5.7 kDa. A polypeptide with the characteristics of the eukaryotic subunit III is missing, and N,N-dicyclohexyl-carbodiimide, a specific reagent for this component, labels subunit I. Under controlled conditions and even at physiological pH, the single subunit present at Mr <10000 can be selectively removed from the complex. Hydrophobic photolabeling suggests that with the mitochondrial subunits I and II only subunit IV among the nuclear coded polypeptides is in contact with lipids. © 1985 American Chemical Society. INDEX KEYWORDS: cytochrome c oxidase; dicyclohexylcarbodiimide, dictyostelium discoideum; enzyme subunit; fungus; nonhuman; priority journal CHEMICALS/CAS: cytochrome c oxidase, 72841-18-0, 9001-16-5; dicyclohexylcarbodiimide, 538-75-0