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

• Published in: Journal of cellular biochemistry Vol. 47; no. 2; p. 147
• Main Author: Robichon, A
• Format: Journal Article
• Language: English
• Published: United States 01.10.1991
• Subjects: 3',5'-Cyclic-GMP Phosphodiesterases - antagonists & inhibitors; 3',5'-Cyclic-GMP Phosphodiesterases - isolation & purification; 3',5'-Cyclic-GMP Phosphodiesterases - metabolism; Animals; Blood Platelets - enzymology; Catalysis; Cattle; Chromatography, Affinity; Cyclic AMP - metabolism; Cyclic GMP - metabolism; Dipyridamole - pharmacology; Electrophoresis, Polyacrylamide Gel; Kinetics; Phosphorylation; Platelet Activation; Protein Kinases - metabolism; Purinones - pharmacology; Substrate Specificity
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.240470208

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.