Effects of cations on affinity of calmodulin for calcium: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes. Theoretical approach to study of multiple ligand binding to a macromolecule

The acid stability of calmodulin has been used to devise a rapid and efficient method of decalcification based on trichloroacetic acid precipitation. Study of the competitive binding of K+, Mg2+, and Ca2+ to the Ca2+-binding sites of calmodulin has allowed determination of the intrinsic binding constants of each of the three cations for the four Ca2+-binding sites. The data are compatible with an ordered binding of Ca2+. If the Ca2+ sites are labeled A, B, C, and D starting at the NH2 terminus, the order of binding is postulated to be B, A, C, and D. The ordered binding properties support the suggestion that calmodulin translates quantitative Ca2+ signals into qualitatively different cellular responses.