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Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

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Summary

A vanadate-sensitive H+-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H+-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H+ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H+ transport activity was inhibited half-maximally between 1 and 5 μM vanadate (Na3VO4) and nearly abolished by 100 μM vanadate. ATPase activity of native plasma membrane showed aK i for vanadate inhibition of 9.5 μM, and was inhibited up to 80% by 15 to 20 μM vanadate (Na3VO4). ATPase activity of the reconstituted vesicles showed aK i of 2.6 μM for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.

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  1. Section of Plant Biology, Division of Biological Sciences, Cornell University, 14853, Ithaca, New York

    Sharman D. O'Neill & Roger M. Spanswick

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  1. Sharman D. O'Neill
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  2. Roger M. Spanswick
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O'Neill, S.D., Spanswick, R.M. Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris . J. Membrain Biol. 79, 231–243 (1984). https://doi.org/10.1007/BF01871062

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  • DOI: https://doi.org/10.1007/BF01871062

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