Large-scale purification and phosphorylation of a detergent-treated adenosine triphosphatase complex from plasma membrane of Saccharomyces cerevisiae.
A new procedure for large-scale preparation of plasma-membrane-bound ATPase from Saccharomyces cerevisiae is described. The crude membrane fraction is purified by selective extraction with three successive detergents: deoxycholate (0.25 mg/mg protein), Triton X-100 (0.25%) and lysophosphatidylcholine (1 mg/mg protein). These treatments extract the mitochondria and strip the plasma membrane. From 1 kg commercial baker's yeast, 200 mg of plasma membrane proteins are isolated in 2--3 days. Plasma-membrane-bound ATPase of specific activity of 10--13 mumol Pi x min-1 x mg protein-1 is obtained with a yield estimated to 60%. Dodecylsulfate/polyacrylamide gel electrophoresis shows three predominant polypeptides of Mr = 95000, 70000 and 56000 in the purified membrane fraction. The major polypeptide of Mr = 95000 identified as the ATPase subunit is phosphorylated by millimolar concentrations of ATP. The phosphorylated intermediate reaches the steady-state level in less than 100 ms and turns over very rapidly. It is hydrolyzed by hydroxylamine. Its formation is prevented by the ATPase inhibitors vanadate and Dio-9, a plasma-membrane ATPase inhibitor of unknown structure. At least four other membrane proteins are phosphorylated with much slower kinetics, presumably through the action of protein-kinase(s).
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003840:Deoxycholic Acid; D003902:Detergents; D007700:Kinetics; D008244:Lysophosphatidylcholines; D046911:Macromolecular Substances; D008970:Molecular Weight; D017830:Octoxynol; D010766:Phosphorylation; D011092:Polyethylene Glycols; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003902:Detergents; D008244:Lysophosphatidylcholines; D046911:Macromolecular Substances; D003840:Deoxycholic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol; D000251:Adenosine Triphosphatases