Evidence for voltage-sensitive reaction steps in the mechanism of the red beet plasma membrane H+-ATPase

The effects of an imposed voltage on the ATP hydrolytic and transport activities of the red beet (Beta vulgaris L.) plasma membrane H+-ATPase were examined following its purification and reconstitution into proteoliposomes or a planar bilayer membrane. When a large opposing (interior-positive) membrane voltage was established in proteoliposomes, ATP hydrolytic activity of the plasma membrane H+-ATPase decreased, but its sensitivity to vanadate was also reduced. Although an opposing voltage decreased ATP-dependent electrical current generation by the H+-ATPase, little change in the vanadate sensitivity of this activity was observed. These results were interpreted in terms of a voltage-sensitive reaction occurring at the E1P → E2P transition step in the enzyme reaction mechanism. The close fit of current-voltage (I/V) data to a two-state reaction-kinetic model for the red beet plasma membrane H+-ATPase, in a planar bilayer or proteoliposomes, was also consistent with the presence of a voltage-sensitive step in the mechanism being involved in charge translocation. Moreover, direct measurement of rate constants for the E1P → E2P reaction based upon dephosphorylation kinetics confirmed voltage-sensitivity for this step. The results of this study are discussed in terms of a role for the E1P → E2P reaction step in H+ translocation and the possibility of the plant plasma membrane H+-ATPase displaying “slip” in the presence of an opposing membrane voltage.