Purification and characterization of a human platelet cyclic nucleotide phosphodiesterase

Abstract

A cyclic nucleotide phosphodiesterase was extensively purified from the 100000g supernatant fraction of human platelets. The purification was 2500-3000-fold with 30% recovery of activity. The enzyme was isolated by DEAE-cellulose chromatography followed by adsorption to blue dextran-Sepharose and elution with cAMP. The protein has a molecular weight of 140 000 as determined by gel filtration. On NaDodSO4-containing polyacrylamide gels the major band is at 61 000 daltons, suggesting that the enzyme may exist as a dimer in solution under nondenaturing conditions. The enzyme requires Mg2+ or Mn2+ for activity. The calcium binding protein calmodulin does not stimulate hydrolysis of cAMP by this enzyme. The purified enzyme hydrolyzes both cAMP and cGMP with normal Michaelis-Menten kinetics with Km values of 0.18 microM and 0.02 microM, respectively. The hydrolysis of cGMP, however, is only one-tenth as rapid as the hydrolysis of cAMP. Cyclic GMP does not stimulate cAMP hydrolysis but instead is a potent competitive inhibitor of cAMP hydrolysis. The enzyme is also competitively inhibited by the phosphodiesterase inhibitors papaverine, 3-isobutyl-l-methylxanthine, and dipyridamole. The enzyme did not cross-react with an antibody raised to a cAMP phosphodiesterase isolated from dog kidney, indicating that the enzymes are not immunologically related. The inhibition of cAMP hydrolysis by cGMP suggests a possible regulatory link between these two cyclic nucleotides. One of the roles of cGMP in platelets may be to potentiate increases in intracellular cAMP by inhibiting the hydrolysis of cAMP by this enzyme.

Distinctive anatomical patterns of gene expression for cGMP-inhibited cyclic nucleotide phosphodiesterases.

“…Type III PDEs (PDE3s), commonly referred to as cGMPinhibited cyclic nucleotide phosphodiesterases ( 11), have been isolated from a number of tissues, including adipose tissue ( 12), platelets (13), myocardium (14), vascular (15,16) and tracheo-bronchial (17,18) smooth muscle preparations, liver (19,20), placenta (21), and lymphocytes (22). PDE3s are characterized by a high affinity for cAMP and cGMP, with the V,,.…”

Differential expression of cGMP‐inhibited cyclic nucleotide phosphodiesterases in human hepatoma cell lines

“…cGMP is a competitive inhibitor of cAMP hydrolysis by PDE3 [6][7][8][9][10][11][12][13][14]. Inhibition of cAMP hydrolysis by cGMP also distinguishes PDE3s (cGI PDE) from PDE4s (cAMP-specific PDE) which exhibit a 'low Km' for cAMP and are inhibited by rolipram but not by cGMP [1].…”

“…Most of the platelet PDE3 is cytosolic [12,13,27,28], whereas in adipocytes and hepatocytes, PDE3 activity is predominantly particulate [2,6,7,9,10,29,30] and in heart, both particulate and cytosolic [31][32][33][34]. In HepG2 and Hep3B most of the PDE3 activity was in the particulate fraction, but in HuH7 30% of PDE3 activity was in the supernatant fractions (Fig.…”

“…The PDE3 or cGMP-inhibited (cGI) PDE family is characterized by a high affinity for cAMP and cGMP (with Vm~x greater for cAMP than for cGMP) and competitive inhibition of its cAMP hydrolytic activity by cGMP, cilostamide (OPC 3689) and certain positive inotropic agents, including milrinone and enoximone [6]. PDE3s have been purified from adipose and cardiac tissues, rat liver, bovine aortic smooth muscle and human platelets [6][7][8][9][10][11][12][13]. Studies with specific PDE3 inhibitors suggest that PDE3s regulate cAMP pools important in myocardial contractility, platelet aggregation, vascular smooth muscle relaxation, lipolysis and vascular smooth muscle and T-lymphocyte proliferation [1][2][3][4][5][6][14][15][16][17][18][19].…”

A Sensitive Assay of Human Blood Platelet Cyclic Nucleotide Phosphodiesterase Activity by HPLC Using Fluorescence Derivatization and Its Application to Assessment of Cyclic Nucleotide Phosphodiesterase Inhibitors.

“…Human platelets contain 3 forms of PDE which differ with respect to substrate specificity; PDE I hydrolyzes cAMP and cGMP, equally, PDE III has a higher affinity for cAMP than cGMP, and PDE V is cGMPspecific. [1][2][3][4][5][6] Recently, highly specific inhibitors for each of the PDE isoforms have been developed for therapeutic uses. These inhibitors which elevate cAMP and/or cGMP level(s) of the platelets and inhibit platelet aggregation and adehesion have therapeutic possibilities such as a drug for angina treatment and thrombosis.…”