The biphasic active transport of calcium by the fragmented sarcoplasmic reticulum as revealed by the flow dialysis method.

The calcium accumulated by the fast uptake has an apparent association constant of 0.8 X 10(6) M-1 and a maximum of 80 nmol/mg protein. The fast uptake and the initial rate of the slow uptake show a similar dependence on the calcium concentration when the latter ranges from 5 to 50 muM. The fast uptake is a linear and the slow uptake rate an exponential function of the reticulum concentration. Both uptakes of calcium display a fast and nearly total isotopic equilibration between intra- and extravesicular calcium. After depletion of ATP the calcium accumulated during the slow uptake is released, while that sequestered during the fast uptake is retained by the vesicles, though it remains rapidly exchangeable. After depletion of ITPor acetylphosphate, or addition of EGTA, the release is more substantial, but is almost complete only after addition of ionophore X537A or deoxycholate. The presence of oxalate strongly reduces the rates of these releases. It is concluded that in the steady state observed after the depletion of ATP, a Ca2+ gradient exists through the sarcoplasmic membrane, and the sarcoplasmic pump works at equilibrium. The fast uptake is an active transport and not an active binding. The slow uptake represents an extension of the calcium capacity of the vesicles due to the phosphate liberated by the sarcoplasmic ATPase.