Effects of intracellular free Ca and rate of Ca influx on the Ca pump

The activity of the Ca pump of human red blood cells was studied in resealed ghosts as a function of intracellular free Ca (fCai). Resealed ghosts were made by the agarose column method to contain, in addition to other constituents, less than 0.1 microM fCai, 100 microM arsenazo III, and either 1 mM ATP plus an ATP regenerating system (active ghosts) or no added ATP and no regenerating system (passive ghosts). The rate of Ca influx into these ghosts was manipulated by suspending them in solutions containing various combinations of free Ca (1-30 microM) and the Ca ionophore A23187 (0.1-0.7 microM). Entering Ca increased the fCai and stimulated the pump in active ghosts. In passive ghosts, all the Ca movement could be described by a single rate constant. The activity of the Ca pump was calculated from the rate of net Ca uptake in the active ghosts, using the rate constant for passive Ca movement as determined in the passive ghosts. fCai and the rates of Ca transport in both active and passive ghosts were calculated from the absorbance of entrapped arsenazo III. In general, increasing fCai from 1 to 10 microM activated the pump. Higher fCai caused an inhibition compared with peak activity. The maximum rate of pumping was 80 microM/min. The major new finding is that the rate of active transport at a given fCai appeared to vary with the rate of fCai accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)