H + -Pumping Driven by the Vanadate-Sensitive ATPase in Membrane Vesicles from Corn Roots

Abstract

The initial rate of quenching of quinacrine fluorescence was used to monitor Mg:ATP-dependent H(+)-pumping in membrane vesicles from corn (Zea mays L. cv WF9 x MO17) roots and obtain a preparation in which vanadate-sensitive H(+)-pumping could be observed. Separation of membranes on a linear sucrose density gradient resulted in two distinct peaks of H(+)-pumping activity: a major one, at density 1.11 grams per cubic centimeter, was sensitive to NO(3) (-) and resistant to vanadate, while a minor one, at density 1.17 grams per cubic centimeter, was substantially resistant to NO(3) (-) and sensitive to vanadate. A membrane fraction enriched in the vanadate-sensitive H(+)-pump could be obtained by washing microsomes prepared in the presence of 10% glycerol with 0.25 molar KI. The kinetics of inhibition of H(+)-pumping by vanadate in this membrane preparation indicated that most of the H(+)-pumping activity in this fraction is sensitive to inhibition by vanadate, 50% inhibition being reached at about 60 micromolar vanadate. This value is fairly close to that observed for inhibition by vanadate of the ATPase activity in similar experimental conditions (40 micromolar). The inhibitor sensitivity, divalent cation dependence, pH optimum (6.5), and K(m) for ATP (0.7 millimolar) of the H(+)-pumping activity match quite closely those reported for the plasma membrane ATPase of corn roots and other plant materials.