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

Abstract

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 aKi 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 aKi 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.