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Articles

H⁺-ATPase Activity from Storage Tissue of Beta vulgaris¹

II. H⁺/ATP Stoichiometry of an Anion-Sensitive H⁺-ATPase

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

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 (A₃₄₀) or by monitoring the release of ³²P from [-³²P]ATP. Inasmuch as this anion-sensitive H⁺-ATPase is strongly inhibited by NO₃^–, initial rates of H⁺ influx and ATP hydrolysis were measured in the absence and presence of NO₃^– to account for ATPase activity not involved in H⁺ transport. The NO₃^–-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.

¹ Supported by United States Department of Agriculture Competitive Grants Program grant 81-CRCR-1-0758.

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