Large‐Scale Purification and Phosphorylation of a Detergent‐Treated Adenosine Triphosphatase Complex from Plasma Membrane

Abstract

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 (0.025mg/mg protein), Triton X‐100 (0.25%) and lysophosphatidyclcholine (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 μmol P_i× min ⁻¹× mg protein ⁻¹is obtained with a yield estimated to 60%. Dodecylsulfate/ polyacrylamide gel electrophoresis shows three predominant polypeptides of M_r= 95000, 70000 and 56000 in the purified membrane fraction. The major polypeptide of M_r= 95000 identified as the ATPase submit 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).