Determination of H+/ATP Stoichiometry for the Plasma Membrane H+-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

Abstract

The H(+)/ATP stoichiometry was determined for the plasma membrane H(+)-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H(+)/ATP stoichiometry utilized a kinetic approach where rates of H(+) influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H(+) influx were based upon either determining the initial rate of alkalinization of the external medium from pH 6.13, measuring the rate of vesicle H(+) leakage from a steadystate pH gradient after stopping the H(+)-ATPase or utilizing a mathematical model which describes the net transport of H(+) at any given point in time. When the rate of H(+) influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H(+)/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (DeltaG(atp)), this value for H(+)/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo.