Lumenal Ca2+ dissociation from the phosphorylated Ca(2+)-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 KC1), 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.
Forge
V
V
Département de Biologie Cellulaire et Moléculaire, Centre d'Etudes de Saclay, Gif-sur-Yvette, France.
Mintz
E
E
Canet
D
D
Guillain
F
F
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*
D002413:Cations, Divalent; D010750:Phosphoproteins; D011189:Potassium Chloride; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium