Proton transport activity of the purified vacuolar H+-ATPase from oats: Direct stimulation by Cl-

John M. Ward, Heven Sze

Research output: Contribution to journalArticle

40 Scopus citations

Abstract

To determine whether the detergent-solubilized and purified vacuolar H+-ATPase from plants was active in H+ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K+-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K+ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H+ pumping was electrogenic. Both H+ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A1, a specific vacuolar type ATPase inhibitor. The reconstituted H+ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO3- but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H+ pumping by the reconstituted ATPase in the presence of K+ and valinomycin. Hence, our results support the idea that the vacuolar H+-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl- concentration. The purified and reconstituted H+-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H+ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H+ translocation.

Original languageEnglish (US)
Pages (from-to)925-931
Number of pages7
JournalPlant physiology
Volume99
Issue number3
DOIs
StatePublished - Jan 1 1992

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