Partial purification of adenosine 3′,5′-cyclic monophosphate phosphodiesterases from rat pancreas in the presence of excess protease inhibitors

(i) Three forms of cyclic AMP phosphodiesterases (3′,5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17), F1, F2-I and F2-II, were partially purified from the soluble fraction of rat pancreas in the presence of excess protease inhibitors by DEAE-cellulose column chromatography and gel filtration and were characterized. (ii) F2-II, which was purified 31-fold, exhibited a single peak of activity on both polyacrylamide-gel electrophoresis and isoelectric focusing. The enzyme had a molecular weight of about 70,000, an isoelectric point of 3.9, and an optimal pH around 8.5 and required Mg 2+ or Mn 2+ but not Ca 2+ for activity. The K m values of this enzyme for cyclic AMP and cyclic GMP were 1 and 50 μ m, respectively, while V values of this enzyme for cyclic AMP and cyclic GMP were 36.1 and 12.6 nmol min −1 (mg of protein) −1, respectively. Cyclic GMP competitively inhibited hydrolysis of cyclic AMP by this enzyme. Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] also inhibited hydrolysis of cyclic AMP competitively, with a K i value of 1 μ m. (iii) Fraction F1, which was purified 10-fold, had a molecular weight of more than 500,000 and required Mg 2+ for activity. Its K m values for cyclic AMP were 1 and 5 μ m. Its K m value for cyclic GMP was 45 μ m. Fraction F2-I, which was purified 26-fold, had a molecular weight of about 70,000. The ratio of the initial velocity of hydrolysis of cyclic GMP to that of cyclic AMP was 0.5 at a substrate concentration of 1 μ m.