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Thermodynamics of Cation Binding to Rabbit Skeletal Muscle Calsequestrin

Cap+ binding to rabbit skeletal calsequestrin was studied at physiological ionic strength by equilibrium flow dialysis, Hummel-Dryer gel filtration and microcalorimetry. 31 Cap+-binding sites with a mean dissociation constant (KO) of 0.79 mM were titrated in the absence, and 23 sites with a Kn of 0.88 mM in the presence of 3 mM Mg'+. No cooperativity was observed. For Mg"+ binding, the combination of gel filtration and microcalorimetry yielded a stoichiometry of 26 Mg"+/ protein with a K D of 2 mM. 1 mM Ca2+ decreased the stoichiometry to 20 Mg2+/protein. Binding of Ca2+ in the absence and presence of 3 mM Mg2+ was accompanied by a release of 2.0 and 2.7 H+/protein, respectively. Mg2+ binding did not lead to a significant proton release suggesting a qualitative difference in the Ca"+and Mg'+-binding sites. After correction for proton release, the enthalpy change for Ca"+ binding was very low (-1.5 kJ/protein in the absence, and -15 kJ/protein in the presence of 3 mM Mg2+). The entropy change (+59 J/K.site in the absence and +56 J/K.site in the presence of Mg"+) was therefore virtually the sole driving force for Ca"+ binding. Mg"+ binding is slightly more exothermic (-12.6 kJ/protein), but, as for Ca"+, the entropy change (+50 J/K.site) constituted the major driving force of the reaction. A fluorimetric study indicates that the conformation of tryptophan in Mg2+saturated calsequestrin was clearly different from that in the Ca'+-saturated protein, but that the (Caz+ + Mg"+)-saturated protein was not distinct from the Ca"+saturated protein. Thus, in addition to the thermodynamic characterization of the Ca"+/calsequestrin interaction, our data indicate that Ca"+ and Mg"+ do not bind to the same sites on calsequestrin. The data also predict considerable proton fluxes upon Ca"+-Mg"+ exchange in vivo.