| Summary: | 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 of === Graduate
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