Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells

Ca2+-transport and its energy consumption were studied in intact human red cells loaded with Ca2+ by the aid of the ionophore A23187. After the complete elimination of the ionophore the passive Ca2+-permeability of the membrane returned to its normal low value, except when the intracellular Ca2+-concentration was higher than 3 mM or the ATP level fell below 100 muM. Within these limits the rate of Ca2+-extrusion was independent of the cellular ATP content but was greatly enhanced by increasing [Ca2+]i and reached a plateau at about 1 mM intracellular Ca2+-concentration. The maximum rate of Ca2+-efflux was about 85 mumol/l of cells per min at 37 degrees C, pH 7.4. The activation energy of active Ca2+-extrusion was found to be 15 200 cal/mol, and the optimum pH in the suspension was 7.7. Ca2+-efflux was not connected with the counter-transport of cations. The Ca2+-pump was not affected by ouabain or oligomycin and only partial inhibition could be achieved by the SH-reagents: ethacrynic acid, N-ethylmaleimide and p-chloromercuribenzoate or with propranolol and ruthenium red. An 80 to 95% inhibition of the active Ca2+-extrusion was brought about by 50-250 muM lanthanum, which in the above concentrations caused no aggregation or haemolysis. The inhibition of the Ca2+-pump by lanthanum was found to be reversible, the site of inhibition being at the external surface of the cell membrane. To examine the energy consumption of the Ca2+-extrusion, ATPase activity was assessed by measuring inorganic phosphate liberation in Ca2+-loaded red cells the metabolism of which was inhibited by iodoacetamide + Na+-tetrathionate. Ca2+-activated ATPase activity connected with the Ca2+-pump was distinguished from other Ca2+-ATPases by using the non-penetrating inhibitor, lanthanum. The molar ratio of Ca2+-transported per ATP split was found to be 2 : 1.