Partial characterization of a phosphorylated intermediate associated with the plasma membrane ATPase of corn roots

Abstract

The phosphorylated protein associated with a deoxycholate-extracted plasma membrane fraction from corn (Z. mays L. var. WF9 .times. Mol7) roots was characterized in order to correlate its properties with those of plasma membrane ATPase. Its phosphorylation, like that of plasma membrane ATPase, was dependent on Mg2+, substrate specific for ATP, insensitive to azide, oligomycin or molybdate, and sensitive to N,N''-dicyclohexylcarbodiimide, diethylstilbestrol or vanadate. Monovalent cations affected the phosphorylation of the protein in a manner consistent with their stimulatory effects on ATPase. For K+, this was shown to occur through an increase in the turnover of the phosphoenzyme. Analysis of the phosphorylated protein by NaDodSO4[sodium dodecyl sulfate]/polyacrylamide gel electrophoresis revealed the presence of a single labeled polypeptide with a MW of .apprx. 100,000. Phosphorylation of this polypeptide was dependent on Mg2+, sensitive to K+ and inhibited by vanadate. It is concluded that this polypeptide represents the catalytic subunit of the plasma membrane ATPase. These results are discussed in terms of a model for the coupling of metabolic energy to H+ and K+ transport in higher plants.