Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris |
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Authors: | Sharman D. O'Neill Roger M. Spanswick |
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Affiliation: | (1) Section of Plant Biology, Division of Biological Sciences, Cornell University, 14853 Ithaca, New York |
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Abstract: | 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 M vanadate (Na3VO4) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aKi for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na3VO4). ATPase activity of the reconstituted vesicles showed aKi of 2.6 M 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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Keywords: | H– -ATPase plant plasma membrane solubilization reconstitution vanadate red beet |
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