Effect of Ca and Calmodulin on DeltapH Formation in Tonoplast Vesicles from Corn Roots

The effects of calmodulin (CaM) on ATPase activity and ATP-dependent formation of a proton gradient (DeltapH) were studied in tonoplast membrane vesicles from corn (Zea mays L.) roots. At 0.6 micromolar, CaM stimulated ATPase activity by about 20% in the absence of an uncoupler, but by only 4% in its presence. Thus, the uncoupler-dependent increment of activity was decreased 30 to 45% by CaM. The formation of a proton gradient across the membrane vesicle, measured by quinacrine fluorescence quench, was inhibited about 20% by CaM. Its effect was additive to the effect of Ca(2+) and was completely abolished by EGTA. These effects of CaM could be due to stimulation of H(+) efflux or due to inhibition of the H(+)-ATPase. To distinguish between these possibilities, we examined the effect of CaM on dissipation of preformed DeltapH after the ATPase was inhibited. CaM stimulated the dissipation of a preformed DeltapH by 40% after the H(+)-ATPase was inhibited with NO(3) (-). This indicates that CaM facilitates the recycling of protons across the tonoplast membranes and does not regulate the H(+)-ATPase by direct inhibition.