Purification and kinetic properties of two soluble forms of calmodulin-dependent cyclic nucleotide phosphodiesterase from rat pancreas.
The calmodulin-dependent cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase (EC 3.1.4.17) activity of rat pancreas was purified 280-fold by affinity chromatography on calmodulin-Sepharose 4B. It then accounted for 15% of the total cytosol cyclic GMP nucleotide phosphodiesterase activity, in the presence of Ca2+, and represented a minor component of proteins specifically adsorbed by the column. This activity was resolved on a DEAE-Sephacel column into two fractions, termed PI and PII, on the basis of their order of emergence. After this step, PI and PII were purified 5650- and 3700-fold respectively. The molecular weight of PI was 175 000 and that of PII was 116 000, by polyacrylamide-gradient-gel electrophoresis. Both forms of phosphodiesterase could hydrolyse cyclic AMP and cyclic GMP, although PII displayed a higher affinity toward cyclic GMP than toward cyclic AMP. PI and PII exhibited negative homotropic kinetics in the absence of calmodulin. Upon addition of calmodulin, both enzymes displayed Michaelis-Menten kinetics and a 5-9-fold increase in maximal velocity, at physiological concentrations of cyclic GMP and cyclic AMP. When a pancreatic extract freshly purified by affinity chromatography was immediately analysed by high-performance gel-permeation chromatography on a TSK gel G3000 SW column, PII represented as much as 78% of the eluted activity. This percentage decreased to 52% when the sample was stored at 0 degrees C for 20 h before analysis, suggesting that PII, possibly predominant in vivo, was converted into the heavier PI form upon storage.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002846:Chromatography, Affinity; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D010179:Pancreas / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D015106:3',5'-Cyclic-GMP Phosphodiesterases