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

The effects of Mg2+ ion, pH, and KC1 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 KC1 concentration. Cooper-ativity between the Ca2+-binding sites was observed, and increased with decreasing pH or increasing KC1 concentration. Free Ca2+ ion concentration was decreased by adding MgCl2 ion at low Mg2+ concentration and increased at higher concentra-tions 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. Calmodulin was reported at first as a Ca2+-regulat- by this protein. In order to understand fully th