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.

Abstract Bovine heart cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase requires a heat-stable protein activator for full enzymic activity. Enzyme preparations, largely freed of the protein activator, possess low enzyme activity which is independent of Ca2+. Addition of excess protein activator stimulates the enzyme activity 6- to 10-fold. This activation by the protein activator is shown to be completely dependent on the presence of low concentrations of Ca2+. The concentration of Ca2+ required to give 50% of the maximal activation is 2.3 µm. An equilibrium binding study has shown that 45Ca 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 µm, respectively. The results suggest that the complex of Ca2+ and the protein activator is the true activator for cAMP phosphodiesterase.