Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two‐phase partitioning

Plasma membrane vesicles were isolated from the roots of 7‐day‐old rice plants (Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two‐phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate‐sensitive K+, Mg2+‐ATPase activity that was 18 times higher in the upper (PEG‐rich) phase than in the lower (Dextran‐rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 mM KCl and 3 mM ATP with apparent Km (ATP) and Vmax of 0.75 mM and 79 μmol (mg protein)−1 h−1, respectively. With 50 mM KCl and 7 mM ATP a pH optimum of 6.5, an apparent Km (ATP) of 6.3 mM and Vmax of 159 μmol (mg protein)−1 h−1 were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca2+ but insensitive to azide and nitrate.