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

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 x 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 mumole/ml. cells.min at 28 degrees 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) mumole/ml. cells. Transport from cells containing 5.10(-7) mumole/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 mumole 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 mumole/ml. cells) about 50% of the total is free Ca(2+) on account of binding mainly to dialysable cell constituents.