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+/PPi stoichiometry was determined to be 1 using methods I and II. Experimental limitations did not permit an analysis of the H+/PPi, 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.
ASJC Scopus subject areas
- Molecular Biology