Mitochondrial Adenosinetriphosphatase from Yeast,Saccharomyces cerevisiae.Purification, Subunit Structure and Kinetics

Abstract

A procedure for the purification of ATPase extracted by chloroform from baker''s yeast (S. cerevisiae) is reported. The yield based on submitochondrial particles was 55% and the purification was 100-fold. The isolated complex was homogeneous as assessed by gel filtration, ion-exchange chromatography, sedimentation in sucrose gradient and in the analytical ultracentrifuge. The MW determined by gel filtration was 400,000 .+-. 20,000. Ultracentrifugation yielded .**GRAPHIC**. = 12.50 .+-. 0.13 S and the laser light scattering study gave a diffusion coefficient of D20,w = 2.92 .times. 10-7 cm2 s-1. The amino acid composition, absorption, fluorescence and circular dichroism spectra, from which the helicity of 39% was evaluated, are given. On polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, 6 components with MW of 58,500 (.alpha.), 55,000 (.beta.), 42,000, 34,000 (.gamma.), 10,000 (.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., .gamma. and .delta. and .epsilon. components. The maximum specific activity of the enzyme was 200 U[units]/mg under the optimum conditions. The enzyme was inactivated by incubation at 0.degree. C and strongly inhibited by the antibiotic Dio-9 but not by oligomycin and N,N''-dicyclohexylcarbodiimide. The effects of kinetic parameters and anions on the enzyme are reported. Two active sites for Mg-ATP with Km values of 0.045 mM and 0.37 mM and a single active site for Mg-ITP with Km = 0.450 mM and for Mg-GTP with Km = 0.179 mM 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 kJ/mol).