V. Forge et al., LUMENAL CA2-ATPASE OF THE SARCOPLASMIC-RETICULUM IS SEQUENTIAL( DISSOCIATION FROM THE PHOSPHORYLATED CA2+), The Journal of biological chemistry, 270(31), 1995, pp. 18271-18276
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.