title: The two calcium ions initially bound to nonphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase can no longer be kinetically distinguished when they dissociate from phosphorylated ATPase toward the lumen.
abstract: Using rapid filtration, we investigated the kinetics of release toward the lumen of sarcoplasmic reticulum vesicles of the two Ca2+ ions transported by the Ca(2+)-dependent ATPase of these vesicles. Release rates at 20 degrees C were measured by three methods, with vesicles previously made leaky with an ionophore. First, we measured the rate at which 45Ca2+ bound to ATPase approached its steady-state level after addition of ATP to the 45Ca(2+)-equilibrated ATPase. At pH 6 in the absence of potassium, the observed kinetics did not reveal any very fast phase of 45Ca2+ dissociation from phosphorylated ATPase. Second, we measured the kinetics of 45Ca2+ dissociation from phosphorylated ATPase in a "chase" experiment, by isotopic dilution of calcium under turnover conditions in the presence of potassium. We found that these kinetics were essentially monophasic. Moreover, when they were measured in the presence of a high concentration of calcium, designed to saturate the low-affinity calcium transport sites on the lumenal side of the ATPase, they only departed slightly from monophasic behavior, irrespective of the experimental pH (pH 6, 7, or 9). This small perturbation by high calcium concentrations of the observed dissociation kinetics was attributed to ADP-facilitated rapid exchange of 40Ca2+ for Mg2+ at the catalytic site of phosphorylated ATPase. The third method was based on the fact that phosphorylation-induced 45Ca2+ occlusion occurred faster than 45Ca2+ dissociation from nonphosphorylated ATPase: here, we measured the rate of 45Ca2+ internalization on addition to 45Ca(2+)-saturated ATPase of an unlabeled ATP-containing medium. This method allowed separate observation of the dissociation kinetics of each of the two 45Ca2+ ions bound to phosphorylated ATPase, after either one or the other had been labeled by a preliminary partial isotopic exchange in the non-phosphorylated state of the ATPase. We found that after ATP-induced phosphorylation, the two 45Ca2+ ions dissociated toward the lumenal medium with virtually identical rate constants; this was observed under different ionic and pH conditions and also in the presence of a high Ca2+ concentration. As a control, the same partial isotopic exchange procedure allowed us to confirm that, in contrast, when ATP was absent from the final dissociation medium, the two 45Ca2+ ions dissociated from nonphosphorylated ATPase toward the cytoplasmic medium at different rates, the one bound more deeply only dissociating after a lag period corresponding to dissociation of the superficial one.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Département de Biologie Cellulaire et Moléculaire, Service de Biophysique des Proteins et des Membranes et URA Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
authors.forename: S
authors.initials: S
authors.lastname: Orlowski
authors.forename: P
authors.initials: P
authors.lastname: Champeil
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D008961:Models, Structural; D009132:Muscles / Q000201:enzymology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013997:Time Factors
chemical_list: D002132:Calcium Radioisotopes; D010750:Phosphoproteins; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium