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 (ΔG_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.