Kinetics of Ca/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris L

Abstract

Artificial pH gradients across tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were used to study the kinetics of a Ca(2+)/H(+) antiport across this membrane. Ca(2+)-dependent H(+) fluxes were measured by the pH-dependent fluorescence quenching of acridine orange. DeltapH-dependent Ca(2+) influx was measured radiometrically. Both H(+) efflux and Ca(2+) influx displayed saturation kinetics and an identical dependence on external calcium with apparent K(m) values of 43.9 and 41.7 micromolar, respectively. Calcium influx was unaffected by an excess of Mg(2+) but was inhibited by La(3+) > Mn(2+) > Cd(2+). The apparent K(m) for external calcium was greatly affected (5-fold) by internal pH in the range of 6.0 to 6.5 and a transmembrane effect of internal proton binding on the affinity for external calcium is suggested.