Phosphorylated intermediate of the ATPase from the plasma membrane of yeast

A purified preparation of the plasma-membrane ATPase from Saccharomyces cerevisiae was phosphorylated when incubated with [gamma-32P]ATP. The phosphoprotein formed has the characteristics of an enzyme intermediate because of its rapidity of phosphorylation and dephosphorylation. When the phosphorylated enzyme was analyzed by polyacrylamide gel electrophoresis in sodium dodecylsulfate only one band with a molecular weight of 100000 contained radioactivity. This band represented about 80% of the protein of the preparation and its enrichment in the course of the purification correlated with the increase in the specific ATPase activity. Both the ATPase reaction and the phosphorylation of the enzyme exhibited an apparent dissociation constant for the enzyme-ATP complex of 0.2 mM, further implicating the phosphoenzyme as an intermediate of the reaction. The sensitivity of the phosphoenzyme bond to alkaline pH and hydroxylamine indicate that it is an acylphosphate. From the maximum level of intermediate (0.7 nmol/mg) and the maximum ATPase activity at 30 degrees C (21 mumol x min-1 x mg-1) a turnover number of 30000 min-1 can be calculated. The level of phosphoenzyme was not affected by either the ATPase inhibitors vanadate and dicyclohexylcarbodiimide or by ADP. These results indicate that the yeast plasma-membrane ATPase has a subunit composition and reaction mechanism similar to the cation-pumping ATPases of animal plasma membranes.