Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.1 (phosphodiesterase)
 18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate. 216 Feb 72

Treatment of intact human platelets with the adenylate cyclase agonist forskolin (100 microM) resulted in an increase in cAMP phosphodiesterase activity in freeze-thaw lysates. When the low-Km (high affinity), cGMP-inhibited cAMP phosphodiesterase was isolated from such lysates by blue dextran-Sepharose chromatography, the specific activity of the enzyme was increased an average of 11-fold over similarly processed control platelets. The increase in the low-Km, cGMP-inhibited cAMP phosphodiesterase activity was inhibited when platelets were incubated with the protein kinase inhibitor H-8 prior to treatment with forskolin, suggesting that the stimulation of cAMP phosphodiesterase activity involved a cAMP-dependent phosphorylation. When intact platelets that had been prelabeled with 32Pi were treated with forskolin and the low-Km, cGMP-inhibited phosphodiesterase was isolated by blue dextran-Sepharose chromatography, a protein of 110,000 kDa was phosphorylated. By using a monospecific antiserum to the purified phosphodiesterase, this protein was shown to be the low-Km, cGMP-inhibited cAMP phosphodiesterase by electrophoretic transfer blot (Western blot) analysis and by immunoprecipitation. The stable prostacyclin analog iloprost also stimulated the low-Km cAMP phosphodiesterase activity about 2-fold and caused phosphorylation of the enzyme. These results suggest that phosphorylation of the low-Km, cGMP-inhibited phosphodiesterase may be an important regulatory mechanism for this enzyme in platelets.
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PMID:cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity. 246 61

The positive inotropic action of the newer cardiotonic phosphodiesterase inhibitors such as indolidan, milrinone, and imazodan has been previously attributed to selective inhibition of cGMP-inhibitable Type IV (high affinity) cAMP phosphodiesterase activity. However, the subcellular binding site(s) for this class of compounds has not been defined. We have characterized the binding of [3H]LY186126, an analogue of indolidan, in subcellular fractions prepared from rabbit and sheep ventricular myocardium. Binding required magnesium ion and exhibited rapid association and dissociation kinetics. Specific binding (defined by ligand displacement with 5 microM indolidan) to enriched rabbit sarcoplasmic reticulum (SR) membrane vesicles was saturable (Bmax = 714 +/- 77 fmol/mg of protein) and of high affinity (Kd = 6.2 +/- 1.4 nM). Linear and nonlinear analyses of the binding isotherms fit a single-site model. Mixed SR preparations from sheep myocardium exhibited binding characteristics (Bmax = 944 +/- 115 fmol/mg; Kd = 8.5 +/- 2.3 nM) comparable to those of rabbit cardiac SR. Further subfractionation of sheep SR indicated that the binding sites were equally distributed between free (Bmax = 630 fmol/mg; Kd = 4.4 nM) and junctional SR (Bmax = 569 fmol/mg; Kd = 10.9 nM). Specific binding of [3H]LY186126 was also demonstrated in the cytosolic subfraction of rabbit myocardium that contained Type IV phosphodiesterase activity (Peak III from anion exchange chromatography). Competition for [3H] LY186126 binding studied in rabbit SR showed that, of the compounds tested, lixazinone (RS 82856) competed most effectively (IC50 = 0.030 +/- 0.008 nM), followed by indolidan (0.14 +/- 0.05 nM), cGMP (17.8 +/- 2.6 nM), milrinone (39.3 +/- 13.2 nM), and imazodan (192 +/- 73 nM). In contrast, rolipram, which does not inhibit SR-associated Type IV phosphodiesterase activity, was not effective at competing for [3H]LY186126 binding (IC50 greater than 30 microM). These results indicate that [3H]LY186126 has specific binding sites in myocardial subcellular fractions that contain cGMP-inhibitable Type IV (high affinity) cAMP phosphodiesterase activity.
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PMID:Analysis of the binding sites for the cardiotonic phosphodiesterase inhibitor [3H]LY186126 in ventricular myocardium. 250 59