Purification and Properties of Rat‐Liver‐Mitochondrial Adenosine Triphosphatase

Abstract

Mitochondrial ATPase from rat liver has been highly purified to a maximum specific activity of 110 in Tris‐bicarbonate buffer at pH 8.0. Sedimentation velocity studies in the analytical ultracentrifuge show that the enzyme sediments as a single symmetrical peak with a sedimentation coefficient ( s o 20, w ), of 12.9 S. Molecular weight determinations by both high‐speed sedimentation equilibrium and polyacrylamide gel chromatography indicate a molecular weight of 360000 ± 10000. Polyacrylamide gel electrophoresis of the enzyme dissociated by sodium dodecyl sulfate reveals one major and three minor bands with molecular weights of 53000, 28000, 12500, and 8000–9000. A calculated molecular weight of approximately 365000 is obtained by assuming the presence of six subunits of molecular weight 53000 and one of each of the lighter subunits. Like the beef‐heart enzyme, rat‐liver ATPase activity is lost on incubation at 5°C and examination by high‐speed sedimentation equilibrium at 5°C indicates that the molecule is extensively dissociated to components of low molecular weight. The enzymatic activities of both rat‐liver‐mitochondrial and beef‐heart‐mitochondrial ATPases are sensitive to the anion of the buffer used at pH 8.0 with activity decreasing in the following series: HCO 3 , − > maleate > chloride > acetate = sulfate. Sulfite, chromate and dinitrophenol stimulate the activity of either enzyme in Tris‐sulfate buffer, but only sulfite increases the activity seen in Tris‐bicarbonate buffer. Aurovertin decreases the enzymatic activity to approximately the same level, regardless of the anion(s) present. The enzyme is insensitive to oligomycin.