Ca2+ binding to sarcoplasmic reticulum ATPase revisited. I. Mechanism of affinity and cooperativity modulation by H+ and Mg2+.
H+ and Mg2+ are known to inhibit Ca2+ binding to the transport sites of sarcoplasmic reticulum-ATPase. Evaluation of the affinity for the Ca2+ binding sites requires measurement of the amount of Ca2+ bound to ATPase as a function of the free Ca2+ concentration imposed by a Ca2+ chelator. The choice of the chelator is crucial as it determines the accuracy of the free Ca2+ concentration. At pH > 7, the EGTA affinity for Ca2+ is higher than that of ATPase, inducing artifacts that alter the shape of the binding curves. Thus, we have used 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whose affinity is unchanged at pH > or = 7. Ca2+ binding was studied at equilibrium, from pH 6 to pH 8 and from 0 to 10 mM Mg2+, using EGTA and/or BAPTA and [45Ca]Ca2+. Under all conditions, the stoichiometry was 2 Ca2+/ATPase. At variance with previous studies, the Hill coefficient was 1.1-2 and higher at pH 6 than at pH 8. In addition, it decreased in the presence of Mg2+. The Ca2+ binding curves were analyzed according to a model in which they result from a sequential binding of two Ca2+, each binding step being modified by H+ and Mg2+. The effect of H+ is described by two steps involving two H+ and one H+, with pK 7 and 7.9, respectively. At pH 6, ATPase must lose two H+ for the first Ca2+ to bind and a third H+ for the second Ca2+ to bind. At pH 9, both Ca2+ bind without any H+ exchange. Mg2+ can bind to all species, except to that saturated with Ca2+. The species having lost two H+ has a higher affinity for Mg2+ (< or = 1 mM) than the species having bound three H+ (4 mM). The above model allows us to analyze the effects of H+ and Mg2+ at each Ca2+ binding step and to explain the changes in the apparent affinity and cooperativity.
Centre National de la Recherche Scientifique 1290, Département de Biologie Cellulaire et Moléculaire, Centre d'Etudes de Saclay, Gif-sur-Yvette, France.
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008433:Mathematics; D008954:Models, Biological; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases; D008274:Magnesium; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium