H-ATPase Activity from Storage Tissue of Beta vulgaris: II. H/ATP Stoichiometry of an Anion-Sensitive H-ATPase

Abstract

The H(+)/ATP stoichiometry was determined for an anion-sensitive H(+)-ATPase in membrane vesicles believed to be derived from tonoplast. Initial rates of proton influx were measured by monitoring the alkalinization of a weakly buffered medium (pH 6.13) following the addition of ATP to a suspension of membrane vesicles of Beta vulgaris L. Initial rates of ATP hydrolysis were measured in an assay where ATP hydrolysis is coupled to NADH oxidation and monitored spectrophotometrically (A(340)) or by monitoring the release of (32)P from [gamma-(32)P]ATP. Inasmuch as this anion-sensitive H(+)-ATPase is strongly inhibited by NO(3) (-), initial rates of H(+) influx and ATP hydrolysis were measured in the absence and presence of NO(3) (-) to account for ATPase activity not involved in H(+) transport. The NO(3) (-)-sensitive activities were calculated and used to estimate the ratio of H(+) transported to ATP hydrolyzed. These measurements resulted in an estimate of the H(+)/ATP stoichiometry of 1.96 +/- 0.14 suggesting that the actual stoichiometry is 2 H(+) transported per ATP hydrolyzed. When compared with the reported values of the electrochemical potential gradient for H(+) across the tonoplast measured in vivo, our result suggests that the H(+)-ATPase does not operate near equilibrium but is regulated by cellular factors other than energy supply.