A Calcium‐Dependent Protein Activator of Guanosine 3′:5′‐Monophosphate Phosphodiesterase in Bovine and Rat Pancreas

A protein activator for guanosine 3′:5′‐monophosphate phosphodiesterase from bovine pancreas was purified in high yield and to apparent homogeneity as judged by standard polyacrylamide gel and sodium dodecylsulfate polyacrylamide gel electrophoresis. The purification required heat treatment, ammonium sulfate precipitation, ion‐exchange chromatography on DEAE‐cellulose and gel filtration on Sephadex G‐75. The minimum molecular weight of the activator calculated from the amino acid composition was 18779. Acid hydrolysates contained a high content of aspartic and glutamic acids, a high phenylalanine to tyrosine ratio, and one ɛ‐ N ‐trimethyllysine per mole protein. No cysteine and tryptophan were detected in the protein. The soluble supernatant fraction of rat pancreas homogenate subjected to gel filtration on Sephadex G‐200 revealed 3 peaks (P1, P2 and P3) with cyclic nucleotide phosphodiesterase activity and one peak of the protein activator. P1, P2 and P3 phosphodiesterases were all active to varying extent on both cyclic GMP and cyclic AMP. The activity of P3 with cyclic GMP as substrate was specifically stimulated three–five‐fold by the purified bovine pancreatic protein activator in the presence of free Ca 2+ . Phosphodiesterases P1 and P2 were not stimulated by this activator and were not inhibited by the Ca 2+ ‐chelating agent, EGTA. A comparison of the phosphodiesterase activity of a crude rat pancreas extract in the presence of Ca 2+ or EGTA, showed that 42% of low‐ K m cyclic GMP phosphodiesterase activity was responsive to the Ca 2+ ‐activator complex while cyclic AMP phosphodiesterase was not. In addition, a large excess of protein activator appeared to exist in the rat pancreas.