H+ PPI STOICHIOMETRY OF A MEMBRANE-BOUND PYROPHOSPHATASE OF PLANT-MITOCHONDRIA/

The H+/PPi stoichiometry of the mitochondrial H+-PP(i)ase from pea (Pi sum sativum L.) stem was determined by two kinetic approaches, and com pared with the H+/substrate stoichiometries of the mitochondrial H+-AT Pase, and the vacuolar H+-PP(i)ase and H+-ATPase. Using sub-mitochondr ial particles or preparations enriched in vacuolar membranes, the rate s of substrate-dependent H+-transport were evaluated: by a mathematica l model, describing the time-course of H+-gradient (Delta pH) formatio n; or by determining the rate of H+-leakage following H+-pumping inhib ition by EDTA at the steady-state Delta pH. When the H+-transport rate s were divided by those of PPi or ATP hydrolysis, measured under ident ical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H+-PP(i)ase and H+-ATPase, and for the vacuolar H+-A TPase. The stoichiometry of the vacuolar H+-PP(i)ase was found to be c a 1. From these results, it is suggested that the mitochondrial H+-PP( i)ase may, in theory, function asa primary H+-pump poised towards synt hesis of PPi and, therefore, acting in parallel with the main H+-ATPas e.