EVIDENCE FOR VOLTAGE-SENSITIVE REACTION STEPS IN THE MECHANISM OF THERED 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+-ATPas e were examined following its purification and reconstitution into pro teoliposomes or a planar bilayer membrane. When a large opposing (inte rior-positive) membrane voltage was established in proteoliposomes, AT P hydrolytic activity of the plasma membrane H+-ATPase decreased, but its sensitivity to vanadate was also reduced. Although an opposing vol tage decreased ATP-dependent electrical current generation by the H+-A TPase, little change in the vanadate sensitivity of this activity was observed. These results were interpreted in terms of a voltage-sensiti ve 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+-A TPase, in a planar bilayer or proteoliposomes, was also consistent wit h the presence of a voltage-sensitive step in the mechanism being invo lved in charge translocation. Moreover, direct measurement of rate con stants for the E1P --> E2P reaction based upon dephosphorylation kinet ics confirmed voltage-sensitivity for this step. The results of this s tudy are discussed in terms of a role for the E1P --> E2P reaction ste p in H+ translocation and the possibility of the plant plasma membrane H+-ATPase displaying ''slip'' in the presence of an opposing membrane voltage. (C) Elsevier, Paris.