Phosphorylated intermediate of a transport ATPase and activity of protein kinase in membranes from corn roots.

A maize-root microsomal fraction was enriched in ATPase by treatment with Triton X-100. This activity, which reached 1.2-2.0/mumol Pi x min-1 x mg protein-1, was specific for ATP, very slightly stimulated by K+, inhibited by orthovanadate and diethylstilbestrol, resistant to oligomycin and azide, and had a Km of 1.2 mM MgATP. Incubation of the microsomal fraction with [gamma 32-P]ATP followed by electrophoresis in acid conditions revealed the presence of several phosphoproteins. The phosphorylation of a 110000-Mr polypeptide reached the steady-state level in less than 5 s and rapidly turned over the phosphate group. The phosphorylation level was an hyperbolic function of the [ATP] with a Km of 0.6 mM, suggesting that the rate of Pi production was proportional to the phosphoprotein concentration. The extent of phosphoprotein was decreased by vanadate and diethylstilbestrol. The phosphorylation level was 30% decreased by 50 mM K+ or Na+ while the ATPase activity was slightly stimulated (12% and 5%, respectively). The polypeptide could not be phosphorylated in reverse by Pi. This phosphorylated intermediate from maize-root microsomes exhibits molecular properties characteristic of transport ATPases such as the yeast plasma membrane H+-translocating ATPase. This similarity indicates existence of a transport ATPase in plant plasma membranes. Three other plant microsomal polypeptides (Mr = 52000, 17000 and 16000) and a low molecular weight component (Mr less than 1000) were phosphorylated much more slowly, were not undergoing a rapid turnover and were not hydrolysed by hydroxylamine. These phosphoproteins and the Mr less than 1000 phosphorylated component were inhibited by vanadate and diethylstilbestrol. These properties are similar to those of the protein kinase activity recently described in yeast plasma membranes.