Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from bovine tissues.
A cGMP-stimulated cyclic nucleotide phosphodiesterase has been purified to near homogeneity from bovine adrenal and heart tissues. The purification procedure utilizes chromatography on DEAE-cellulose and cGMP affinity resin. The procedure can be completed within 2 days and is easily adapted to large scale. To obtain pure enzyme, an 8,000-9,000-fold increase in specific activity was required in adrenal tissues and 15,000-30,000-fold in cardiac muscle. A single band of protein having an apparent Mr = 105,000-107,000 was seen on sodium dodecyl sulfate gel electrophoresis. At equilibrium, native polyacrylamide gradient gel electrophoresis revealed a single major band having an apparent Mr = 240,000. Cyclic GMP binding and phosphodiesterase activity co-migrated with the protein band on native polyacrylamide gradient gels. The enzyme bound cGMP with high affinity reaching a maximum binding of 1.02 mol of cGMP bound/mol of enzyme dimer. Titration curves of the binding data indicated at least two classes of binding sites with 10% maximal binding occurring at 7 nM and 90% maximal binding at 4 microM cGMP. Kinetic analysis indicated the enzyme can hydrolyze both cAMP and cGMP with similar maximal rates. The nucleotide concentration at half-maximal velocity were 30 and 10 microM for cAMP and cGMP, respectively. The hydrolyses of both nucleotides exhibit positive homotropic cooperativity with Hill coefficients of 1.9 for cAMP and 1.3 for cGMP. The rate of cAMP hydrolysis by the purified enzyme when measured at 10 microM cAMP was enhanced 5- to 6-fold by low levels of cGMP.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification* / Q000378:metabolism; D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D000242:Cyclic AMP; D006152:Cyclic GMP / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D009928:Organ Specificity; D013379:Substrate Specificity
D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP