Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae.

摘要: Abstract H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially membrane H+-ATPase, which had a specific activity 18 units/mg protein, separated almost completely acid phosphatase alkaline phosphatase. The enzyme required phospholipids for maximal hydrolyzed ATP, GTP, UTP, CTP, this order preference. Its Km value Mg2+-ATP determined to be 0.21 mM its optimal pH 6.9. ADP inhibited competitively, Ki 0.31 mM. ATPase strongly N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, quercetin, but not affected oligomycin, sodium azide, vanadate, or miconazole. It at all antiserum against mitochondrial F1-ATPase inhibitor protein. These results indicated that H+-ATPase is different either yeast plasma F1-ATPase. found composed two major polypeptides b Mr = 89,000 64,000, respectively, N,N'-dicyclohexylcarbodiimide binding polypeptide c 19,500, whose composition also those S. cerevisiae.