title: Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots.
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 K(m) for this ion was 4 millimolar. Fluoride inhibited both DeltapH formation and K(+)-dependent PPase activity with an I(50) of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H(+)-ATPase (e.g. a disulfonic stilbene or NO(3) (-)) 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 Mg(2+) and pyrophosphate (PPi) concentrations. At 5 millimolar Mg(2+), H(+) pumping showed a K(m(aPP) ) 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.
authors: Department of Botany, University of Maryland, College Park, Maryland 20742.
authors: Y
authors: Y
authors: Wang
authors: R A
authors: RA
authors: Leigh
authors: K H
authors: KH
authors: Kaestner
authors: H
authors: H
authors: Sze