Properties of the vanadate-sensitive ATPase of the plasma membrane vesicles from rice moots

The plasma membrane vesicles were purified by aqueous two phase partitioning from rice roots (Oryza sativa L. cv. Nipponbare). The ATPase activity of the purified plasma membrane fraction (U3 fraction) increased more than 4-fold by the addition of Triton X-100, and was not inhibited by trypsin digestion pretreatment. Vanadate inhibited 70% of the ATPase activity of the Triton-washed U3 fraction. The K m value was 1.45 mM, and vanadate and ADP inhibited the activity uncompetitively and competitively, respectively. The ATPase activity was dependent on Mg2+, and Co2+ could be completely substituted for Mg2+, while Fe2+ and Mn2+ partially. KCl increased the activity by 20% with saturation at 5 mM. The order of the stimulation by monovalent cations was NH4 +>K+>Li+, choline+>Na+. The optimum pH for the activity was 6. The vesicles of the U3 fraction were reconstituted with deoxycholate and soybean phospholipids. The proton pumping activity of the fraction was inhibited by vanadate. Based on the results obtained, it was concluded that vanadate-sensitive ATPase, which is presented on the plasma membrane of rice roots, acts as a proton pump.