LUMENAL CA2+ DISSOCIATION FROM THE PHOSPHORYLATED CA2+-ATPASE OF THE SARCOPLASMIC-RETICULUM IS SEQUENTIAL

Once two radioactive Ca2+ coming from the cytoplasm are bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca2+ only reaches one of the two bound Ca2+. This difference has been explained assuming that the two Ca2+ sites are in a single file channel in which the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is exchangeable only when the superficial site is vacant, The same experiment was done using phosphorylated ATPase to determine whether Ca2+ dissociation toward the lumen is sequential as well, Under conditions that allow ADP-sensitive phosphoenzyme to accumulate (leaky vesicles, 5 degrees C, pH 8, 300 mM KCl), we found the same two pools of Ca2+. Excess EGTA induced dissociation of both ions together with dephosphorylation, Excess nonradioactive Ca2+ induced the exchange of half the radioactive Ca2+ without any effect on the phosphoenzyme level, Our results show a close similarity between the transport sites of the nonphosphorylated and the phosphorylated enzymes, although the orientation, affinities, and dissociation rate constants are different.