Purification and Properties of the Protein Activator of Bovine Heart Cyclic Adenosine 3′,5′-Monophosphate Phosphodiesterase

Abstract A protein activator for cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterase has been purified from bovine heart by a simple procedure which involves ammonium sulfate and acid precipitations, heat treatment, DEAE-cellulose and Sephadex G-100 column chromatography. The purified activator appears to be homogeneous by ultracentrifugal and disc gel electrophoretic criteria. The specific activity of the pure activator is approximately 8,000-fold that of the crude tissue extract. For optimal recovery of the activator, the buffer solutions used during purification should contain 1 mm Mg2+. The activator is relatively stable in low concentrations of Mg2+, 1 mm or lower. At higher concentrations of this metal ion, 10 mm or higher, the activator rapidly loses its activity during storage at 4°. The activator is also inactivated by low concentrations of EDTA. This inactivation is not reversed by the addition of Mg2+. Kinetic studies indicate that the activator activates cAMP phosphodiesterase by enhancing the Vmax and decreasing the Km for cAMP. The cyclic nucleotide, in turn, enhances the affinity of the enzyme toward the activator. The dependence of the enzyme activity on the protein concentration of a mixture of the enzyme and the activator is nonlinear, showing upward curvature. These results suggest that the activator and the enzyme may exist in equilibrium among their respective free forms and the active protein complex and that this equilibrium may be modulated by cAMP. The molecular weight of the activator has been determined by sedimentation-diffusion and gel filtration techniques to be 19,200 and 27,000, respectively.