Calcium binding to calmodulin. cooperativity of the calcium-binding sites

The effects of Mg²⁺ ion, pH, and KCl concentration on Ca²⁺ binding to calmodulin were studied by using a Ca²⁺ ion-sensitive electrode. The Ca²⁺ ion affinity of calmodulin increased with increasing pH or decreasing KCl concentration. Cooperativity between the Ca²⁺ sites was observed, and increased with decreasing pH or increasing KCl concentration. Free Ca²⁺ ion concentration was decreased by adding MgCl₂ ion at low Mg²⁺ concentration and increased at higher concentrations in the presence of small amounts of Ca²⁺ ion. The decrease of free Ca²⁺ ion concentration by Mg²⁺ ion strongly suggests cooperativity between the Ca²⁺-binding sites, and it is difficult to explain the decrease in terms of the ordered binding models previously proposed. These results can be explained by a simple model which has four equivalent binding sites that bind Ca²⁺ and Mg²⁺ competitively, and showing cooperativity when either Ca²⁺ or Mg²⁺ is bound. Mg²⁺ ion binding to calmodulin was measured in the presence or absence of Ca²⁺ to confirm the validity of this model, and no Mg²⁺-specific site was observed.