Electrogenic H+-Pumping Pyrophosphatase in Tonoplast Vesicles of Oat Roots

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Abstract
A H+-translocating inorganic pyrophosphatase (H+-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H+-PPase catalyzed the electrogenic transport of H+ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb+ = K+ > Cs+; but it was inhibited by Na+ or Li+. Maximum rates of transport required at least 20 millimolar K+ and the Km for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K+-dependent PPase activity with an I50 of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H+-ATPase (e.g. a disulfonic stilbene or NO3−) had no effect on the PPase activity. Vanadate and azide were also ineffective. H+-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H+-PPase and the H+-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H+ pumping activity was strongly affected by Mg2+ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg2+, H+ pumping showed a KmaPP for PPi of 15 micromolar. The rate of H+ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H+-PPase.