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

Once two radioactive Ca2+ coming from the cytoplasm are bound to the t ransport sites of the nonphosphorylated ATPase, excess EGTA induces ra pid 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 whic h the superficial Ca2+ is freely exchangeable from the cytoplasm, wher eas the deeper Ca2+ is exchangeable only when the superficial site is vacant, The same experiment was done using phosphorylated ATPase to de termine whether Ca2+ dissociation toward the lumen is sequential as we ll, Under conditions that allow ADP-sensitive phosphoenzyme to accumul ate (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 toge ther with dephosphorylation, Excess nonradioactive Ca2+ induced the ex change of half the radioactive Ca2+ without any effect on the phosphoe nzyme level, Our results show a close similarity between the transport sites of the nonphosphorylated and the phosphorylated enzymes, althou gh the orientation, affinities, and dissociation rate constants are di fferent.