Catalytic and regulatory properties of two forms of bovine heart cyclic nucleotide phosphodiesterase

The soluble supernatant fraction of bovine heart homogenates may be fractionated on a DEAE cellulose column into two cyclic nucleotide phosphodiesterases (EC 3.1.4.-): P_I and P_II phosphodiesterases, in the order of emergence from the column. In the presence of free Ca²⁺, the P_I enzyme may be activated several fold by the protein activator which was discovered by Cheung ((1971) J. Biol. Chem. 246, 2859-2869). The P_II enzyme is refractory to this activator, and is not inhibited by the Ca²⁺ chelating agent, ethylene glycol bis (β-aminoethyl ether)-N,N′-tetraacetate (EGTA). The activated activity of P_I phosphodiesterase may be further stimulated by imidazole or NH₄⁺, and inhibited by high concentrations of Mg²⁺. These reagents have no significant effect on either the P_II enzyme or the basal activity of P_I phosphodiesterase. Although both forms of phosphodiesterase can hydrolyze either cyclic AMP or cyclic GMP, they exhibit different relative affinities towards these two cyclic nucleotides. The P_I enzyme appears to have much higher affinities toward cyclic GMP than cyclic AMP. K_m values for cyclic AMP and cyclic GMP are respectively 1.7 and 0.33 mM for the non-activated P_I phosphodiesterase; and 0.2 and 0.007 mM for the activated enzyme. Each cyclic nucleotide acts as a competitive inhibitor for the other with K_i values similar to the respective K_m values. In contrast with P_I phosphodiesterase, P_II phosphodiesterase exhibits similar affinity toward cyclic AMP and cyclic GMP. The apparent K_m values of cyclic AMP and cyclic GMP for the P_II enzyme are approx. 0.05 and 0.03 mM, respectively. The kinetic plot with respect to cyclic GMP shows positive cooperativity. Each cyclic nucleotide acts as a non-competitive inhibitor for the other nucleotide. These kinetic properties of P_I and P_II phosphodiesterase of bovine heart are very similar to those of rat liver cyclic GMP and high K_m cyclic AMP phosphodiesterases, respectively (Russel, Terasaki and Appleman, (1973) J. Biol. Chem. 248, 1334).