Mitochondrial adenosine triphosphatase from yeast, Saccharomyces cerevisiae. Purification, subunit structure and kinetics.
1. A procedure for the purification of ATPase extracted by chloroform from baker's yeast (Saccharomyces cerevisiae) is reported. The yield based on submitochondrial particles was 55% and the purification was 100-fold. The isolated complex was homogenous as assessed by gel filtration, ion-exchange chromatography, sedimentation in sucrose gradient and in the analytical ultracentrifuge. The molecular weight determined by gel filtration was 400000 +/- 20000. Ultracentrifugation yielded s020,w = 12.50 +/- 0.13 S and the laser light scattering study gave a diffusion coeficient of D20w - 2.92 X 10(-7) cm2 s-1. The amino acid composition as well as absorption, fluorescence, and circular dichroism spectra, from which the helicity of 39% was evaluated, are given. 2. On polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, six components with molecular weights of 58500(alpha), 55000 (beta), 42000, 34000 (gamma), 10000(delta), and 8600 (epsilon) were observed with a stoichiometry of 3:3:1:1:1:1. The amino acid composition is given for alpha + beta and gamma as well as delta and epsilon components. 3. The maximum specific activity of the enzyme was 200 U/mg under the optimum conditions. The enzyme was inactivated by incubation at 0 degrees C and strongly inhibited by the antibiotic Dio-9 but not by oligomycin and N, N'-dicyclohexyl-carbodiimide. The effects of kinetic parameters and anions on the enzyme are reported. Two active sites for Mg-ATP with Km values of 0.045mM and 0.37mM and a single activie site for Mg-ITP with Km = 0.179mM were found. A study of the temperature dependence of the maximum activity revealed a straight line in the Arrhenius plots with an activation energy of 11.0 kcal/mol (= 46 kH/mol).
D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000838:Anions; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D009840:Oligomycins / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000648:ultrastructure; D013696:Temperature
D000596:Amino Acids; D000838:Anions; D046911:Macromolecular Substances; D009840:Oligomycins; D000251:Adenosine Triphosphatases