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

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Abstract
1. In resealed human red cells loaded with Ca‐EGTA buffer solutions it was found that the intracellular free Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ concentration and the normal cellular ATP, ADP and Pi 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 Ca2+ on account of binding mainly to dialysable cell constituents.