Characteristics and regulation of active calcium transport in inside-out red cell membrane vesicles.

Sealed, inside-out human red cell membrane vesicles, prepared by a modified method of Steck (Steck T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol 2, pp. 245-281, Plenum Press, New York), accomplish an ATP and Mg2+-dependent uphill calcium uptake with a reproducible maximum rate of 12-15 nmol/mg vesicle protein per min under physiological conditions. This maximum rate is increased by about 60-70% in the presence of a heat-stable cytoplasmic activator protein (calmodulin) obtained from red cells. Calcium efflux from inside-out vesicles is smaller than 0.01 nmol/mg vesicle protein per min at intravesicular calcium concentrations between 0.1 and 20.0 mM. In the presence of Mg2+, active calcium uptake is supported by ATP, ITP, or UTP, but not by ADP, AMP, or p-nitrophenyl phosphate. The optimum pH for the process is 7.4-7.6, and the activation energy is 19-20 kcal/mol, irrespective of the presence or absence of calmodulin. Calcium uptake in inside-out vesicles is unaffected by ouabain or oligomycin, but blocked by low concentrations of lanthanum, ruthenium red, quercetin and phloretin. K+ and Na+, when compared to choline+ or Li+, significantly increase active calcium uptake. This stimulation by K+ and Na+ is independent of that by calmodulin. Concentrated red cell cytoplasm activates calcium uptake at low soluble protein:membrane protein ratios, while a 'deactivation' of the transport occurs at high cytoplasm:membrane protein ratios. A heat-labile cytoplasmic protein fraction antagonizing calmodulin activation, can be separated by DEAE-Sephadex chromatography. Based on these findings the regulation of active calcium transport in human red cells is discussed.