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.
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.
Robichon
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A
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37203.
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
D011688:Purinones; D004176:Dipyridamole; D000242:Cyclic AMP; D011494:Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; C011145:zaprinast; D006152:Cyclic GMP