Characterization of the Effects of Divalent Cations on the Coupled Activities of the H-ATPase in Tonoplast Vesicles.

The substrate requirement of the H(+)-ATPase in purified corn root tonoplast vesicles was investigated. The coupled activities, ATP hydrolysis and proton pumping, were simultaneously supported only by Mg(2+) or Mn(2+). The presence of Ca(2+) or Ba(2+) did not significantly affect the coupled activities. The addition of Cd(2+), Co(2+), Cu(2+), and Zn(2+) inhibited both the hydrolysis of Mg-ATP and the proton transport. However, the inhibition of proton pumping was more pronounced. Based on equilibrium analysis, both ATP-complexed and free forms of these cations were inhibitory. Inhibition of the hydrolysis of Mg-ATP could be correlated to the concentrations of the ATP-complex of Zn. On the other hand, the free Cu(2+) and Co(2+) were effective in inhibiting hydrolysis. For proton pumping, the ATP complexes of Co(2+), Cu(2+), and Zn(2+) were effective inhibitors. However, this inhibition could be further modulated by free Co(2+), Cu(2+), and Zn(2+). While the equilibrium concentrations of Cd-ATP and free Cd(2+) were not estimated, the total concentration of this cation needed to inhibit the coupled activities of the H(+)-ATPase was found to be in the range of 10 to 100 micromolars. The presence of free divalent cations also affected the structure of the lipid phase in tonoplast membrane as demonstrated by the changes of emission intensity and polarization of incorporated 1,6-diphenyl-1,3,5-hexatriene. The differential inhibition caused by these cations could be interpreted by interactions with the protogenic domain of the membrane as previously proposed in "indirect-link" mechanism.