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  • Title: Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl.
    Author: Ward JM, Sze H.
    Journal: Plant Physiol; 1992 Jul; 99(3):925-31. PubMed ID: 16669020.
    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 A(1), a specific vacuolar type ATPase inhibitor. The reconstituted H(+) pump was also inhibited by N,N'-dicyclohexylcarbodiimide or NO(3) (-) 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.
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