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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
Author(s) -
Robichon Alain
Publication year - 1991
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240470208
Subject(s) - phosphodiesterase , pde10a , ibmx , phosphodiesterase 3 , affinity chromatography , biochemistry , chemistry , protein kinase a , enzyme , kinase , thrombin , platelet , phosphorylation , protein subunit , cytosol , binding site , enzyme activator , differential centrifugation , cgmp dependent protein kinase , biology , receptor , mitogen activated protein kinase kinase , forskolin , gene , immunology
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 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 μmol/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.