The kinetics of Ca²⁺ transport and (Ca²⁺ + Mg²⁺)-ATPase activity in human erythrocyte inside-out vesicles

The kinetics and the stoichiometry of the Ca²⁺ pump in human erythrocytes was investigated using inside-out vesicles as a model. Inside-out vesicles were prepared by a modified procedure of Steck and Kant (12) outlined by Quist and Roufogalis(9), Since calmodulin has been shown to regulate the activites of the (Ca²⁺ + Mg²⁺)-ATPase, and the Ca²⁺ transport system (49, 50), its effect on the kinetics and stoichiometry was also investigated in inside-out vesicles treated with EDTA to remove endogeneous calmodulin (78). The calmodulin used in this study was purified from human erythrocytes. The stoichiometry of the Ca²⁺ pump was found to vary with intracellular Ca²⁺ concentration: in a low Ca²⁺ concentration range of 0.71 μM to 24 μM, it was estimated to be close to 2 whereas in the high Ca²⁺ range -50 μM to 300 μM, it was estimated to be close to 1. Calmodulin, ATP concentrations from 0.1 - 3 mM and Mg²⁺ concentrations of 3 mM and above did not alter the stoichiometry while a stoichiometry of 2 was obtained at 1 mM Mg²⁺ concentration at all Ca²⁺ concentrations studied. A model for Ca²⁺ efflux is suggested to explain these results. Calmodulin was found to increase the V[sub max] and the affinity of the (Ca²⁺+ Mg²⁺)-ATPase for Ca²⁺. The high affinity component was found to have a K[sub diss] (K[sub l]) of 2 to 4 μM Ca²⁺ whereas the low Ca²⁺ affinity component was found to have a K[sub diss] of 200 t0 300 μM Ca²⁺ Lowering Mg²⁺concentrations in the presence of calmodulin resulted in the predominance of the high affinity component of the Ca²⁺ - transport system which was inhibited at high Ca²⁺ concentrations. Competition between Ca²⁺ and Mg²⁺ for an inhibitory site located on the high affinity form, E[sub l]P of (Ca²⁺ + Mg²⁺ )-ATPase enzyme is suggested to account for the Ca²⁺ inhibition observed at low Mg²⁺ concentrations.Pharmaceutical Sciences, Faculty ofGraduat