Electrogenic H+-pumping pyrophosphatase in tonoplast vesicles of oat roots

• Published in: Plant physiology (Bethesda) 1986-06, Vol.81 (2), p.497-502
• Main Authors: Wang, Y, Leigh, R.A, Kaestner, K.H, Sze, H
• Format: Article
• Language: English
• Subjects: Adenosine triphosphatases; Anions; ATPASE; AVENA SATIVA; Beets; Biological and medical sciences; Cell physiology; Enzymes; Fluorescence; Fundamental and applied biological sciences. Psychology; HIDROLASAS; HYDROGEN ION; HYDROLASE; HYDROLASES; Oats; Plant physiology and development; Plant roots; Plants; Plasma membrane and permeation; Protons; PYROPHOSPHATASES; RACINE; RAICES; ROOTS; Tonoplast; TRANSLOCACION; TRANSLOCATION
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.81.2.497

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 inbihited 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 $\text{I}_{50}$ 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 $K_{m_{app}}$ 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.