Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells

Abstract

1. In resealed human red cells loaded with Ca‐EGTA buffer solutions it was found that the intracellular free Ca 2+ concentration for half saturation of the Ca transport system (which pumps Ca out of the cell) is equal to or smaller than 4 × 10−6 M and thus closely agrees with the dissociation constant of the Ca + Mg activated membrane ATPase. 2. The maximal rate of Ca transport from resealed cells to medium was found to be 0·148 ± 0·009 μmole/ml. cells.min at 28° C. 3. The rate of Ca transport was unaffected by a variation of the extracellular Ca 2+ concentration from 3·10−7 to 5·10−3 M. 4. Evidence is presented making it probable that the stoichiometric relation between Ca transported and ATP hydrolysed is 1:1 rather than 2:1. 5. As the Ca transport is quite rapid even at half saturation and the passive leak for Ca negligible in intact cells it can be predicted that the steady‐state cellular Ca 2+ concentration must be low, most probably less than 10−6 μmole/ml. cells. Transport from cells containing 5·10−7 μmole/ml. into blood plasma is thermodynamically compatible with the normal plasma Ca 2+ concentration and the normal cellular ATP, ADP and P i content. 6. Treatment with the mercurial PCMBS in the cold for 15 hr allows to load red cells with 1 μmole Ca/ml. cells without destroying their ability to transport Ca after removal of the mercurial. 7. It is shown that at high cellular Ca concentrations (0·1–3 μmole/ml. cells) about 50% of the total is free Ca 2+ on account of binding mainly to dialysable cell constituents.