Calcium transport in human inside-out erythrocyte vesicles.

Inside-out vesicles from human erythrocytes were used to study the kinetics of calcium transport. The amounts of total ATP, MgCl2, and CaCl2 required to yield particular concentrations of MgATP2-, free Mg2+, and free Ca2+ were determined mathematically, and the final concentration of free Ca2+ was confirmed using the calcium indicator dye arsenazo III. Calcium accumulation was linear for 40 min in the presence of ATP and for at least 60 min in the presence of ATP and oxalate. When the calcium ionophore A-23187 was added at the end of an uptake experiment, 95% of accumulated calcium was released. There was a single affinity for calcium with a Km of 3.4 +/- 0.4 microM. Rduced glutathione in the assay medium increased the Vmax but did not alter the Km. The optimal pH was 6.9 to 7.3, and the optimal temperature was 40 degrees C with an Ea of 14.3 kcal/mol. The dependence of calcium accumulation on [MgATP2-] and [ATP4-] could not be measured separately in this assay system. At half-maximal transport rate [MgATP2-] = 1.0 MM and [ATP4-] = 0.1 mM. The data suggest a complex kinetic mechanism.