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

Abstract

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 Ca 2+, 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 Ca 2+ 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 NH 4 +, and inhibited by high concentrations of Mg 2+. 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).