Reconstitution and rapid partial purification of the red beet plasma membrane ATPase

摘要: Red beet (Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution carried out by column chromatography on Sephadex G-200 followed centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the preparation observed when dormant red tissue used extraction/solubilization procedure. Following liposomes, ATP-dependent proton transport could be demonstrated measuring quenching of acridine orange fluorescence. Proton enzyme were inhibited orthovandate but stimulated KNO3. This stimulation most likely results reduction potential generated during electrogenic ATPase. The further characterized found have pH optimum 6.5 presence both Mg2+ K+. specific ATP, insensitive ouabain azide N;N-dicyclohexylcarbodiimide diethylstilbestrol. Stimulation ATP hydrolytic occurred sequence: K+ Rb+ Na+ Cs+ Li+ kinetics non-Michaelis-Menten as membrane-bound forms enzyme. Reconstitution allowed substantial purification resulted enrichment kDa polypeptide representing catalytic subunit.