Reversal of the red beet tonoplast H(+)-ATPase by a pyrophosphate-generated proton electrochemical gradient

The reversal of the tonoplast H(+)-ATPase to mediate ATP synthesis was investigated in tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue. Our approach involved use of the H(+)-PP(i)ase to establish a proton electrochemical gradient (delta muH+) across the tonoplast vesicle membrane to drive the H(+)-ATPase in reverse. However, an initial problem with this approach was the presence of an adenylate kinase activity in the tonoplast fraction that interfered with measurement of ATP synthesis as a coupling between the H(+)-ATPase and H(+)-PP(i)ase. Inclusion of the adenylate kinase inhibitor p1p5-di(adenosine)pentaphosphate (Ap5A) in assays at 50 microM led to a complete inhibition of this activity and allowed measurement of ATP synthesis coupled to PPi hydrolysis. When measured in the presence of Ap5A, PPi-dependent ATP synthesis was blocked by Triton X-100 and inhibited by gramicidin D, imidodiphosphate, nitrate, and bafilomycin A. These results are consistent with PPi-dependent ATP synthesis occurring as a coupled process involving a delta muH+ established across the membrane. Furthermore, the observation that ATP synthesis is inhibited by inhibitors of the tonoplast H(+)-ATPase (nitrate and bafilomycin A) would suggest that this enzyme is involved in the synthetic reaction and can operate in reverse to synthesize ATP from ADP and Pi. A thermodynamic analysis of coupling between the H(+)-PP(i)ase and H(+)-ATPase suggests that PPi-driven ATP synthesis could only occur under these reaction conditions if the H+/substrate stoichiometries for the H(+)-PP(i)ase and H(+)-ATPase were 1 and 2, respectively. These values are consistent with transport stoichiometries previously determined for these enzymes in red beet tonoplast vesicles using kinetic methods.