Studies of soluble rat liver mitochondrial acid ATPases. I. Purification and catalytic properties of ATPase 1.
A method for isolation of a soluble ATPase from rat liver mitochondria after freeze thaw cycling is described. Two enzymatically active fractions were separated by DEAE-cellulose chromatography (ATPase 1 and ATPase 2). ATPase 1 has been purified 300 fold. ATPase 1 was homogenous as judged by polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8-6.0 and the optimum temperature was 45 degrees C. The enzyme follows Michaelis-Menten kinetics: Km (9 X 10(-4) M), Vmax (23,6 mumoles Pi released X min -1 X mg protein -1). The enzyme hydrolysed nucleoside triphosphates, but was inactive upon nucleoside di and monophosphates, glucose 6-phosphate, phosphoserine, pyrophosphate and glycerol 2-phosphate. In contrast to membrane bound ATPase, cations have no effect on the enzyme activity. Nucleoside di and mono phosphates and glycerol 2 phosphate inhibited competitively the enzyme. The enzyme was not affected by oligomycin, but was stimulated by lactate, 2-mercaptoethanol and dithiothreitol.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D005615:Freezing; D005994:Glycerophosphates / Q000494:pharmacology; D007773:Lactates / Q000494:pharmacology; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology
D005994:Glycerophosphates; D007773:Lactates; D000249:Adenosine Monophosphate; D008623:Mercaptoethanol; D000251:Adenosine Triphosphatases; D004229:Dithiothreitol