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/μmol P i × min −1 × gm protein −1 , was specific for ATP, very slightly stimulated by K + , inhibited by orthovanadate and diethylstibestrol, resistant to oligomycin and azide, and had a K m of 1.2 mM MgATP. Incubation of th emicrosomal fraction with [y 32 ‐P]ATP followed by electrophoresis in acid conditions revealed the presence of several phosphoproteins. The phosphorylation of a 110000‐ M 4 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 K m of 0.6 mM, suggesting that the rat e of P i 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 bephosphorylated in reverse by P i . This phosphorylated intermediate from maize‐root microsomes exhibits molecular properties characteristic of transport ATPases such as the yeast plasma membrance H + ‐translocating ATPase. This similarity indicates existence of a transport ATPase in plant plasma membranes. Three other plant microsomal polypeptides ( M r = 52000, 17000 and 16000) and a low molecular weight component ( M r < 1000) were phosphorylated much more slowly, were not undergoing a rapid turnover and were not hydrolysed by hydroxylamine. These phosphoproteins and the M r < 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.