Phosphorylated Intermediate of a Transport ATPase and Activity of Protein Kinase in Membranes from Corn Roots

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Abstract
A maize-root microsomal fraction was enriched in ATPase by treatment with Triton X-100. This activity, which reached 1.2-2.0/.mu.mol Pi .times. min-1 .times. 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 110,000-MW polypeptide reached the steady-state level in < 5 s and rapidly turned over the phosphate group. The phosphorylation level was a 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 (MW = 52,000, 17,000 and 16,000) and a low MW component (MW < 1000) were phosphorylated much more slowly, were not undergoing a rapid turnover and were not hydrolysed by hydroxylamine. These phosphoproteins and the MW < 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.