Cyclic Nucleotide Phosphodiesterases from Rat Anterior Pituitary

Phosphodiesterase activities for adenosine and guanosine 3’ : 5’-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl, to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphodiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1 % Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to CaZ + and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N’-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 105000xg supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 pM substrate being 20 pM and 24 pM for cyclic AMP and 7 pM and 10 pM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent K, values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction I1 enzyme was stimulated 6-7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+.