Mechanism of activation of a cyclic adenosine 3':5'-monophosphate phosphodiesterase from bovine heart by calcium ions. Identification of the protein activator as a Ca2+ binding protein.

largely freed of the protein activator, possess low enzyme activity which is independent of Ca”+. Addition of excess protein activator stimulates the enzyme activity 6to lofold. This activation by the protein activator is shown to be completely dependent on the presence of low concentrations of Ca2+. The concentration of Ca?+ required to give 50% of the maximal activation is 2.3 NM. An equilibrium binding study has shown that “X!a binds to the protein activator. A Scatchard plot exhibits two linear regions suggesting the presence of two sets of Ca2+ binding sites on the protein with different affinities: one high affinity site and two low affinity sites per protein activator. The dissociation constants for Ca2+ bound at the high and low affinity sites are 3 and 12 pM, respectively. The results suggest that the complex of Ca2+ and the protein activator is the true activator for CAMP phosphodiesterase.