Determination of H+/ATP stoichiometry for the plasma membrane H+-ATPase from red beet (Beta vulgaris L.) storage tissue

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 steady-state 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 (delta G(ATP)), this value for H+/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasms membrane in vivo