title: A new cGMP phosphodiesterase isolated from bovine platelets is substrate for cAMP- and cGMP-dependent protein kinases: evidence for a key role in the process of platelet activation.
abstract: The biochemical differences among cGMP phosphodiesterases in platelets have not been thoroughly examined, primarily due to the lack of sufficient purified material. This report describes a simple method developed to isolate a specific bovine platelet cGMP phosphodiesterase. This enzyme is cytosolic in its native form and was purified to an apparent homogeneity by ion-exchange chromatography, affinity chromatography, and density gradient centrifugation. Cyclic GMP binds to a "pseudo-site" when the catalytic site is deprived of Mg++. The affinity for cGMP at alkaline pH in presence of EDTA and IBMX (Kd = 60 nM) suggests that the removal of Mg++ by EDTA converts the catalytic site to a binding site. A ligand affinity chromatography was designed to take advantage of these features. The core enzyme has a molecular weight 190,000 composed of 2 subunits (MW 95,000) and has a specific activity of 2.5 mumol/min/mg. Moreover, this enzyme was phosphorylated by cAMP- and cGMP-dependent protein kinases, suggesting that its activity could be indirectly regulated by cyclic nucleotides. Agents elevating cGMP and cAMP inhibit platelet activation by inhibiting protein kinase C and thrombin induced hydrolysis of phosphatidylinositol 4,5 diphosphate. The antiaggregating properties of some of these agents might therefore be attributed to the fact that they are inhibitors of phosphodiesterases.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37203.
authors.forename: A
authors.initials: A
authors.lastname: Robichon
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002384:Catalysis; D002417:Cattle; D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004176:Dipyridamole / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D010766:Phosphorylation; D015539:Platelet Activation; D011494:Protein Kinases / Q000378:metabolism*; D011688:Purinones / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D011688:Purinones; D004176:Dipyridamole; D000242:Cyclic AMP; D011494:Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; C011145:zaprinast; D006152:Cyclic GMP