Energy Transduction in Tonoplast Vesicles from Red Beet ( Beta vulgaris L.) Storage Tissue: H +/Substrate Stoichiometries for the H +-ATPase and H +-PPase

Abstract

The H +/substrate stoichiometries of the tonoplast H +-ATPase and H +-PPase were determined by a kinetic approach. Using red beet ( Beta vulgaris L.) tonoplast vesicles, rates of substrate-dependent H + transport were estimated by (I) a mathematical model describing the time course of ΔpH formation, (II) the rate of H + leakage following H + pump inhibition at a steady state ΔpH, and (III) the initial rate of alkalinization of the external medium. When compared with rates of substrate hydrolysis measured under identical conditions, all three methods yielded an H +/ATP stoichiometry of 2 while the H +/ PP i stoichiometry was determined to be 1 using methods I and II. Experimental limitations did not permit an analysis of the H +/ PP i, stoichiometry by method III. From these results and the estimated level of substrate and product typically found in the cytoplasm of plant cells, it is suggested that the H +-ATPase and H +-PPase as primary H +-pumps are poised toward net substrate hydrolysis under in vivo conditions thereby operating in parallel to generate a proton electrochemical gradient across the tonoplast.