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

cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) binds tightly to a Zn(2+)-chelate column (Francis, S. H., and Corbin, J. D. (1988) Methods Enzymol. 159, 722-729). Using three different approaches, Zn2+ is now shown to bind to cG-BPDE, and the Kd is determined to be approximately 0.5 microM, with a binding stoichiometry of approximately 3 mol of Zn2+/mol of monomer. A similar concentration range of Zn2+ (0.05-1 microM Zn2+) also supports phosphodiesterase (PDE) catalytic activity. The Zn2+ binding to cG-BPDE is not diminished by, nor is catalysis supported by, relatively high concentrations of Cu2+, Cd2+, Ca2+, or Fe2+. Neither cGMP nor 3-isobutyl-1-methylxanthine affects Zn2+ binding under the conditions used. Mn2+, Co2+, or Mg2+ supports catalysis, but only at significantly higher concentrations (4-, 15-, and 250-fold, respectively) than that required for Zn2+. Two tandem amino acid sequences, which are conserved in the catalytic domains of all characterized mammalian PDEs, resemble the single sequence motif that has been shown to coordinate Zn2+ in the catalytic sites of Zn2+ hydrolases such as thermolysin.
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PMID:Zinc interactions and conserved motifs of the cGMP-binding cGMP-specific phosphodiesterase suggest that it is a zinc hydrolase. 807 92

The search for cyclic nucleotide phosphodiesterase inhibitors in large chemical and natural product libraries is limited by assay throughput. A high-throughput assay that can monitor different phosphodiesterase activities would be useful for these inhibitor searches. We have developed a sensitive phosphodiesterase assay which is based on the selective precipitation of the radiolabeled 5'-nucleotide product by zinc and barium. This assay is unique to previous precipitation methods in that it measures product formed, either GMP or AMP. The procedure was adapted to a microtiter plate format for high-throughput, making it possible to perform in excess of 960 assays per day. This method was used to determine accurate enzyme kinetic constants for three different phosphodiesterases. The results agree favorably with literature values. Using the cyclic guanosine 3',5'-monophosphate-specific phosphodiesterase, IC50 values for the inhibitors M&B 22,948 and 3-isobutyl-1-methylxanthine were also determined. Demonstration of appropriate Km and inhibitor IC50 values verifies the ability of this new assay to identify selective cyclic nucleotide phosphodiesterase inhibitors.
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PMID:A high-throughput assay for cyclic nucleotide phosphodiesterases. 813 46

An Zn(2+)-GPC cholinephosphodiesterase activity, which is present more predominantly in myelin than in microsome or cytosol, has been examined using rho-nitrophenylphosphocholine as a substrate. In the solubilization of enzyme activity from myelin membranes, lysolecithin was found to be more effective than Triton X-100 or deoxycholate. Especially, the myelin-bound phosphodiesterase was suggested to be a glycosylphosphatidyl-inositol-anchored protein, based on solubilization by B. cereus phospholipase C and Triton X-114 phase separation. Interestingly, it was found that while phospholipase C-solubilized enzyme, a hydrophilic protein, was associable with Concanavalin A column, detergent-solubilized amphiphilic form of enzyme was not. Either detergent extract or cytosol was observed to contain both amphiphilic form and hydrophilic one. In CM-sephadex chromatography, the soluble hydrophilic phosphodiesterase was observed to be separatable into two forms of enzyme. In comparative studies, both forms of phosphodiesterase showed much similarity in substrate specificity, optimum pH, Km value and Zn2+ requirement, although they differed in charge property and molecular weight.
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PMID:Brain myelin-bound Zn(2+)-glycerophosphocholine cholinephosphodiesterase is a glycosylphosphatidylinositol-anchored enzyme of two different molecular forms. 813 71

The model of rat primary hepatocytes incubated in DMEM/F12 (Ham) medium was used for studying the influence of the cAMP-effectors epinephrine (100 microM), norepinephrine (100 microM), glucagon (1 microM) and isoproterenol (1-1000 microM) as well as the synthetic cAMP-analogon dibutyryl-cAMP on the metabolism of metallothionein. Liver parenchymal cells isolated by a two-step collagenase perfusion were incubated with DMEM/F12 containing 5% (v/v) fetal calf serum (FCS) and 20 microM zinc in Petri dishes. Experiments were initiated after a 24 h equilibration period by adding the agonists for 18 h. MT in hepatocyte homogenates was quantified by the 109Cd-hemoglobin-binding assay. Cell viability was assessed by the activity of the cytosolic enzyme lactate dehydrogenase (LDH) liberated into the culture medium and by trypan blue exclusion. Isoproterenol and glucagon produced a significant increase of cytosolic MT about 50%. In contrast, incubation with epinephrine and norepinephrine did not lead to any significant effects in the amount of hepatic metallothionein. Simulating the influence of cAMP by dibutyryl-cAMP (500 microM) did not affect the content of hepatic metallothionein. To examine transcriptional and translational regulatory effects supplementation of cycloheximide (0.1-500 microM) and actinomycin D (0.1-100 microM) showed a total inhibition of the agonist induced amounts. Particularly in combination with isoproterenol low LDH activities reflected a high viability of hepatocytes. In conclusion, in primary hepatocyte cultures cAMP-mobilizing-agonists like isoproterenol and glucagon indicate an independent effect on the MT-metabolism. This is possibly due to the de novo synthesis of the protein because suppression by actinomycin D can be observed. However, cAMP-effectors do not seem to be involved in the induction of metallothionein because theophylline and dibutyryl-cAMP did not affect MT-metabolism by suppressing the phosphodiesterase or by stimulating the cAMP-cascade.
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PMID:Influence of cAMP-effector-agonists on the synthesis of metallothionein in rat primary hepatocytes. 858 45

This paper describes the purification and properties of a 2',3'-cyclic nucleotide 3'-phosphodiesterase which hydrolyzes nucleoside 2',3'-cyclic monophosphates to nucleoside 2'-phosphates. The enzyme is present in encysted gastrulae of Artemia and its specific activity greatly increases during larval development. The purified enzyme has a molecular weight of around 55 000 as estimated by gel filtration, does not require metals for activity, is inhibited by Zn2+ and inactivated by Cu2+ and has a pH optimum at around neutrality. Based on the relative values of V(max)/Km, the specificity of the phosphodiesterase toward the four 2',3'-cyclic nucleotides is Guo-2',3'-P > Ado-2',3'-P > Cyd-2',3'-P > Urd-2',3'-P = 45:36:20:7. The enzyme from Artemia gastrulae is competitively inhibited by the four nucleosides 2'-phosphates (Ki values around 1 mM) while the enzyme from larvae is only inhibited by the purine nucleotides. The phosphodiesterase characterized in this work is more similar in substrate specificity to the 2',3'-cyclic nucleotide 3'-phosphodiesterase from the mammalian nervous system than to the plant enzyme. The functional relationship of this enzyme with the Artemia ribonuclease VI is discussed.
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PMID:Purification and characterization of Artemia 2',3'-cyclic nucleotide 3'-phosphodiesterase. 864 16

A Zn(2+)-glycerophosphocholine cholinephosphodiesterase was purified with a specific activity of 4.6 mumole/min.mg protein from bovine brain membranes by procedures involving PI-PLC solubilization, concanavalin A affinity chromatography, CM-sephadex chromatography and Sephadex G-150 chromatography. Based on molecular weight determination gel chromatography and SDS polyacrylamide gel electrophoresis, the phosphodiesterase activity appears to be a dimeric protein (110 kDa) composed of two subunits with a molecular weight of approximately 54 kDa. The K(m) value for p-nitrophenylphosphocholine and the optimum pH were found to be 16 microM and pH 10.5, respectively. The phosphodiesterase was inhibited by Cu2+, but not the other divalent metal ions. The activity of the apoenzyme was remarkably activated by Co2+ or Zn2+, but not Mn2+ or Mg2+. In addition, the inactivation of the enzyme in glycine buffer was prevented by Mn2+ or Zn2+, but not Co2+ or Mg2. In a separate experiment, comparing properties of the purified and membrane-bound phosphodiesterases, the forms of two enzymes were quite similar except in stability. Both enzymes were more stable at pH 7.4 than pH 5 or 10. However, the membrane-bound enzyme was more stable than the soluble enzyme at all three pHs. These data suggest that the activity of the phosphodiesterase may be stabilized in-vivo.
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PMID:Properties of a Zn(2+)-glycerophosphocholine cholinephosphodiesterase from bovine brain membranes. 892 80

A potential role for cAMP in regulating the differentiation of myoblasts has led us to examine the components of the cAMP signaling system, including the type IV, cAMP-specific phosphodiesterases. The full coding sequence of the phosphodiesterase PDE4D1 was inserted in the bacterial expression vector pGEX-KG. N- and C-terminal truncations were also placed in the same vector, allowing the expression and purification of glutathione S-transferase (GST)-PDE fusion proteins using glutathione-Sepharose. The purified PDE was active [V(max) = 318 +/- 18 nmol min(-1)(mg of protein)(-1)] and inhibited by RO 20-1724, rolipram, and MIX (IC50 values of 2, 0.4, and 40 microM, respectively). The requirement of PDE4D1 for a divalent cation was also examined. It was able to use Mg2+, Co2+, and Mn2+, but not Zn2+, suggesting that it is not a zinc hydrolase as has been proposed for other PDE types. Deletion of both C- and N-terminal regions affected the apparent native size of the enzyme. The C-terminal region was involved in dimer formation, whereas an N-terminal region was responsible for larger aggregates. Removal of the last 35 amino acids of an N-terminal 80-residue highly conserved region (UCR2) resulted in a 6-fold increase in PDE activity, providing evidence that this part of the molecule acts as an intramolecular inhibitor. The availability of a highly purified, enzymatically active protein in substantial quantities has allowed us to directly examine PDE4D1 for the first time.
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PMID:Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants. 906 27

A microdialysis method combined with a sensitive radioimmunoassay was used to monitor cGMP release in the frontal cortex of the anesthetized rats in vivo. We assessed the relative contribution of endogenous nitric oxide (NO), and effects of exogenous carbon monoxide (CO) and phosphodiesterase activity, as possible regulators of cortical CGMP levels. Perfusion with CO-saturated aCSF (approximately 1 mM CO) failed to significantly stimulate cortical cGMP levels. For comparison, cerebellar cGMP levels increased by 2-fold during CO stimulation, followed by a prolonged response that was fully reversible with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Cortical perfusion with zinc protopophyrin-IX (100 microM), a widely used inhibitor of the CO-generating enzyme heme oxygenase, suppressed cGMP levels by 50%, a response that spontaneously recovered in spite of the continuous presence of the metalloporphyrin. Perfusion with isobutylmethyl xanthine IBMX (1 mM) resulted in 5-fold increase in cortical cGMP levels, as compared to basal levels without IBMX. In the presence of IBMX, L-NAME suppressed basal cortical cGMP levels by 70% indicating that NO synthase activity generates the bulk of cGMP in this brain region, as previously shown for basal cGMP production in the hippocampus and the cerebellum. These data also emphasize a crucial role for phosphodiesterase activity in the maintenance of cGMP levels in vivo in the frontal cortex. The relatively weak responses to exogenous CO lend little support for a role of this gas in regulating basal cortical cGMP levels in vivo.
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PMID:Regulation of cyclic GMP levels in the rat frontal cortex in vivo: effects of exogenous carbon monoxide and phosphodiesterase inhibition. 917 94

The effect of divalent metal ions on the activity of glycerophosphocholine cholinephosphodiesterse from ox brain was examined. Zn(2+)- and Co(2+)-glycerophosphocholine cholinephosphodiesterases were prepared from the exposure of apoenzyme to Zn2+ and Co2+, respectively, and the properties of two metallo-phosphodiesterases were compared to those of native phosphodiesterase. Although two metallo-enzymes were similar in expressing Km value, optimum pH or sensitivity to Cu2+, they differed in the susceptibility to the inhibition by thiocholine or tellurite; while Co(2+)-phosphodiesterase was more sensitive to tellurites, Zn(2+)-phosphodiesterase was more susceptible to inhibition by thiocholine. In addition, Zn(2+)-phosphodiesterase was more thermo-stable than Co2+ enzyme. Separately, when properties of native phosphodiesterase were compared to those of each metallo-phosphodiesterase, native phosphodiesterase was found to be quite similar to Zn(2+)-phosphodiesterase in many respects. Even in thermo-stability, native enzyme resembled Zn(2+)-phosphodiesterase rather than Co(2+)-enzyme. Consistent with this, the stability of native phosphodiesterase was maintained in the presence of Zn2+, but not Co2+, Mn2+ was also as effective as Zn2+ in the stabilization of the enzyme. Noteworthy, the native enzyme was found to be inhibited competitively by Cu2+ with a Ki value of 20 microM, and its inhibitory action was antagonized effectively by Zn2+ or Co2+. Also, choline, another competitive inhibitor of the enzyme, appeared to antagonize the inhibitory action of Cu2+. Taken together, it is suggested that there may be multiple binding sites for divalent metal ions in the molecule of glycerophosphocholine cholinephosphodiesterase.
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PMID:Interaction of divalent metal ions with Zn(2+)-glycerophosphocholine cholinephosphodiesterase from ox brain. 935 12

The neuroendocrine system plays a key role in the regulation of the secretion of the thymic peptide, thymulin, but it remains to be determined whether thymulin exerts reciprocal regulatory actions on the functional activity of the hypothalamo-pituitary axis. In the present study, we have used a well established in vitro preparation to examine the influence of thymulin on cyclic nucleotide formation and hormone secretion by the rat anterior pituitary gland. Thymulin-Zn2+ (0.5-50 pM) stimulated the release of immunoreactive corticotrophin (ir-ACTH), producing effects which were maximal at 10 pM (p < 0.01). At the two highest concentrations tested (10 and 50 pM), it also produced small but significant increases in immunoreactive luteinising hormone (ir-LH) release (p < 0.05), but the secretion of immunoreactive growth hormone (ir-GH) was unaffected by the peptide (p > 0.05) while that of immunoreactive prolactin (ir-PRL) was reduced (p < 0.01). The ACTH responses to thymulin were accompanied by increased cyclic nucleotide formation. Thus, thymulin (0.5-50 pM) raised the cyclic 3',5'-adenosine monophosphate (cyclic AMP) content of the pituitary tissue (p < 0.01). At high concentrations (10-50 pM), it also increased cyclic 3',5'-guanosine monophosphate (cyclic GMP; p < 0.01) accumulation, although lower concentrations of the peptide were ineffective in this regard. The increases in ir-ACTH release provoked by thymulin-Zn2+ (0.5-5.0 pM) were potentiated markedly by rolipram (1 microM; p < 0.01), a selective inhibitor of the cyclic-AMP-specific phosphodiesterase enzyme. By contrast, zaprinast (10 microM), a selective inhibitor of cyclic-GMP-specific phosphodiesterase, attenuated the corticotrophic responses to higher concentrations of the peptide (10 and 50 pM; p < 0.05). Neither rolipram (1 microM) nor zaprinast (10 microM) influenced the release of ir-LH, ir-PRL or ir-GH in the presence or absence of thymulin-Zn2+ (0.5-50 pM; p > 0.05). The results suggest that thymulin modulates the secretion of ACTH and possibly LH by the anterior pituitary gland and that its actions are associated with increased cyclic nucleotide formation; in addition, it appears to exert an inhibitory influence on ir-PRL release.
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PMID:Thymulin stimulates corticotrophin release and cyclic nucleotide formation in the rat anterior pituitary gland. 948 96


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