Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Endogenous calmodulin (CaM) in the EGTA-washed cerebral-cortical synaptosomal membrane (SM) preparation was estimated below 3 micrograms/ml protein by the semiquantitative immunoblot analysis (Natsukari, N., Ohta, H. and Fujita, M. (1989) J. Immunol. Methods 125, 159-166). Membrane-bound CaM was immunoelectron-microscopically demonstrated in EGTA-washed, non-treated (control), and Ca(2+)-treated cerebral-cortical synaptosomal membranes (SM) as well as for the SM enriched with added CaM. The density of CaM increased in the above order. CaM-dependent adenylate cyclase and CaM-dependent protein kinase II (CaM-kinase II) activities were restored, whereas the phosphodiesterase (PDE) activity was not affected by exogenous CaM over all the Ca2+ concentrations tested. Adenylate cyclase at pCa 6.2 was synergistically activated either by GTP and CaM or by CaM and beta-adrenergic agonist, (+/-)-isoproterenol, reflecting the intactness of signal transduction pathway in the SM. Also demonstrated were the presence of protein kinase A, CaM-kinase II, and their endogenous substrates in the SM. Based on 32P-autoradiography and 125I-CaM overlay data certain CaM-binding proteins such as CaM-kinase II and synapsin I were identified on SDS-PAGE. Ca(2+)-dependent and -independent CaMBPs were distinguished by 125I-CaM gel overlay with and without Ca2+. The former had bigger molecular size (greater than or equal to 49 kDa) than the latter (less than or equal to 34 kDa). Yield of Ca(2+)-dependent CaMBPs was not affected by Ca2+ concentration during preparation of the SM while that of Ca(2+)-independent CaMBPs was reduced by exposure to 100 microM Ca2+. In contrast with the CaMBPs of brain SM, those of enterocyte and eyrthrocyte plasma membranes especially, microvillous membrane of the enterocyte, showed quite distinct CaMBP profiles. The present findings suggested that the EGTA-washed SM preparation made a useful system for studying the role of CaM in the brain SM.
...
PMID:Characterization of EGTA-washed synaptosomal membrane with emphasis on its calmodulin-binding proteins. Demonstration of possible reconstitution with added calcium/calmodulin. 131 53

The mechanism of adenylyl cyclase desensitization by carbachol, an agent that stimulates polyphosphoinositide hydrolysis, was studied in thyroid cells. Incubation of cultured dog thyroid cells with 10 microM carbachol for 2-4 hr reduced the subsequent thyrotropic hormone (TSH) stimulation of adenylyl cyclase activity of membrane preparations by approximately 40%. This inhibition was reversed by atropine, occurred even in a Ca(2+)-free medium containing ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, and was not reproduced by the Ca2+ ionophore A23187. The carbachol effect was not prevented by simultaneous incubation of cells with either isobutylmethylxanthine, an inhibitor of phosphodiesterase, or H-7, an inhibitor of protein kinase. Pretreatment of cells with pertussis toxin to inactivate the Gi inhibitory protein also failed to affect the carbachol inhibition. Although carbachol did not reduce the basal or the TSH-stimulated cyclase activities when added to membranes directly during the assay, exposure of cells to carbachol for 2-4 hr resulted in long lasting inhibition of TSH-stimulated cyclase activity (for at least 24 hr); recovery was seen by 48 hr after its removal. Carbachol pretreatment had no effect on 125I-TSH binding to membranes but reduced the cyclase stimulation by not only TSH but also cholera toxin, guanosine 5'-O-(3-thio)triphosphate, and forskolin; it also significantly reduced the cholera toxin-mediated AD[32P]-ribosylation of Gs in membranes. These data indicate that carbachol-induced inhibition of adenylyl cyclase occurs beyond the level of TSH receptor binding and that Gs is a possible site of its action. Thus, in dog thyroid cells, carbachol, via muscarinic receptors, can reduce the adenylyl cyclase activity by a process that does not involve Ca2+ or activation of phosphodiesterase.
...
PMID:Carbachol-induced decrease in thyroid cell adenylyl cyclase activity is independent of calcium and phosphodiesterase activation. 131 Jan 40

Calmodulin is the major intracellular Ca(2+)-binding protein, providing Ca(2+)-dependent regulation of numerous intracellular enzymes. The phosphorylation of calmodulin may provide an additional mechanism for modulating its function as a signal transducer. Phosphocalmodulin has been identified in tissues and cells, and calmodulin is phosphorylated both in vitro and in intact cells by various enzymes. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreases its ability to activate both myosin-light-chain kinase and cyclic nucleotide phosphodiesterase. For myosin-light-chain kinase the primary effect is an inhibition of the Vmax. of the reaction, with no apparent change in the concentration at which half-maximal velocity is attained (K0.5) for either Ca2+ or calmodulin. In contrast, for phosphodiesterase, phosphorylation of calmodulin significantly increases the K0.5 for calmodulin without noticeably altering the Vmax. or the K0.5 for Ca2+. The higher the stoichiometry of phosphorylation of calmodulin, the greater the inhibition of calmodulin-stimulated activity for both enzymes. Therefore the phosphorylation of calmodulin by casein kinase II appears to provide a Ca(2+)-independent mechanism whereby calmodulin regulates at least two important target enzymes, myosin-light-chain kinase and cyclic nucleotide phosphodiesterase.
...
PMID:Phosphorylation by casein kinase II alters the biological activity of calmodulin. 131 63

The type V cyclic GMP phosphodiesterase was partially purified from the high-speed supernatant of guinea-pig lung. The isoenzyme displayed linear kinetics for cyclic GMP hydrolysis, with Km = 2.2 +/- 0.2 microM and Vmax. = 1.2 +/- 0.08 nmol/min per mg. The selective type V phosphodiesterase inhibitor Zaprinast inhibited cyclic GMP hydrolysis with IC50 (concn. giving 50% inhibition) = 0.45 +/- 0.08 microM. Isobutylmethylxanthine promoted a 3-fold increase in the binding of cyclic GMP to the isoenzyme. The addition of the catalytic subunit of protein kinase A to an activation cocktail containing the partially purified type V phosphodiesterase resulted in a marked increase in Vmax. for cyclic GMP hydrolysis (approximately 10-fold at 40 units of protein kinase A). We have suggested that protein kinase A triggers phosphorylation of the phosphodiesterase, which results in activation of phosphodiesterase activity. In addition, the sensitivity to inhibition by Zaprinast is severely decreased (the IC50 for inhibition is 7.5 +/- 1.1 microM), suggesting that the potency of phosphodiesterase inhibitors is effected by phosphorylation of the enzyme.
...
PMID:The catalytic subunit of protein kinase A triggers activation of the type V cyclic GMP-specific phosphodiesterase from guinea-pig lung. 131 15

Expression of the Epstein-Barr virus (EBV) BZLF1 gene product ZEBRA is a first step in the cascade of the virus-productive cycle. ZEBRA protein was detected by immunoblotting as a single band at 38 kDa in Akata cells after crosslinkage of membrane immunoglobulin G (IgG) with anti-IgG antibody. Immunoprecipitation of [32P]phosphate-labeled, anti-IgG-stimulated Akata cells with anti-ZEBRA antibody showed that ZEBRA was phosphorylated. Phosphoamino acid analysis demonstrated phosphorylation of serine, but not threonine or tyrosine, and tryptic-peptide mapping showed multiple phosphorylated peptides of ZEBRA. Treatment with 8-bromo cAMP and blockage of phosphodiesterase by theophylline in anti-IgG-stimulated cells increased the phosphorylation of three ZEBRA peptides. Incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced the phosphorylation of these three ZEBRA peptides, while treatment with staurosporine, a protein kinase C (PKC) inhibitor, enhanced their phosphorylations. These data suggest that activation of PKC with TPA induces the ZEBRA dephosphorylation and that activation of cAMP-dependent protein kinase A enhances the ZEBRA phosphorylation at the specific sites.
...
PMID:Phosphorylation of the Epstein-Barr virus BZLF1 immediate-early gene product ZEBRA. 131 87

Insulin induced phosphorylation and activation of the cGMP inhibited cAMP phosphodiesterase (cGI-PDE) in human platelets were demonstrated after isolation of the enzyme with specific polyclonal cGI-PDE antibodies. The demonstration of this insulin effect required suppression of basal cGI-PDE phosphorylation, through the use of the protein kinase inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine). The human platelet insulin receptor beta-subunit, previously identified as a 97 kDa polypeptide, was detected with the use of wheat germ agglutinin chromatography and anti-phosphotyrosine antibodies. These results suggest that insulin, through phosphorylation/activation of cGI-PDE, could decrease cAMP/cAMP dependent protein kinase (cAMP-PK) activity and thereby make the platelets more sensitive towards aggregating agents.
...
PMID:Insulin induced phosphorylation and activation of the cGMP-inhibited cAMP phosphodiesterase in human platelets. 132 13

8-(p-Chlorophenylthio)-cGMP (8-pCPT-cGMP) and 8-bromo-cGMP were compared with respect to their chemical and biological properties in order to evaluate their potential as selective activators of cGMP-dependent protein kinase (cGMP-PK; EC 2.7.1.37) in intact human platelets. 8-pCPT-cGMP, 8-Br-cGMP and cGMP were shown to be potent and selective activators of purified bovine lung cGMP-PK and of cGMP-PK present in human platelet membranes when compared with the activation of cAMP-dependent protein kinase (cAMP-PK; EC 2.7.1.37). 8-pCPT-cGMP was not hydrolysed by the purified cGMP-stimulated phosphodiesterase (cGS-PDE), cGMP-inhibited phosphodiesterase (cGI-PDE) and Ca(2+)-calmodulin-dependent phosphodiesterase (CaM-PDE), whereas cGMP and, to a lesser extent, 8-Br-cGMP were hydrolysed by all three types of 3',5' cyclic nucleotide phosphodiesterases (EC 3.1.4.17) examined. Also, 8-pCPT-cGMP was not hydrolysed by a human platelet homogenate which contains a high level of the cGMP-specific cGMP-binding phosphodiesterase (cGB-PDE). Additionally, 8-pCPT-cGMP did not activate the cGS-PDE or inhibit the cGI-PDE, whereas half-maximal inhibition of cGI-PDE occurred at 8 microM 8-Br-cGMP. The apparent lipophilicity of 8-pCPT-cGMP was higher than that of 8-Br-cGMP. Extracellular application of 8-pCPT-cGMP to intact human platelets reproduced the pattern of protein phosphorylation induced by sodium nitroprusside (SNP), a cGMP-elevating inhibitor of platelet activation. Quantitatively, 8-pCPT-cGMP was more effective than 8-Br-cGMP in inducing phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein, a major substrate of cGMP-PK in intact platelets. As observed with SNP, pretreatment of human platelets with 8-pCPT-cGMP prevented the aggregation induced by thrombin. The results suggest that 8-pCPT-cGMP is a very potent and selective activator of cGMP-PK in cell extracts and in intact human platelets and, in this respect, is superior to 8-Br-cGMP and other cGMP analogs used for intact cell studies. The data also suggest that inhibition of platelet activation in intact human platelets by nitrovasodilators is mediated by cGMP-PK.
...
PMID:Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP. 132 24

Autoradiographic localizations of major second messengers and a selective cyclic adenosine monophosphate (cyclic-AMP) phosphodiesterase in the brain were visualized in the gerbil and the rat using receptor autoradiography. [3H]Phorbol 12,13-dibutyrate (PDBu), [3H]inositol 1,4,5-trisphosphate (IP3), [3H]forskolin, [3H]cyclic-AMP, and [3H]rolipram were used to label protein kinase C, IP3 receptor, adenylate cyclase, cyclic-AMP-dependent protein kinase (cyclic-AMP-DPK), and Ca2+/calmodulin-independent cyclic-AMP phosphodiesterase (PDE), respectively. Most second messengers and rolipram binding activities were especially found in the limbic system, basal ganglia, and cerebellum. Marked differences were noted in the hippocampus, where cyclic-AMP and rolipram binding activities were very low in gerbils but high in rats. In contrast, regional localization in the binding sites of PDBu, IP3, and forskolin in gerbil brain was relatively similar to that in rat brain. Further, alteration of the cyclic-AMP and rolipram binding sites was studied in the gerbil hippocampus 7 days after 10-min cerebral ischemia. The results suggest that the gerbil differs from the rat with respect to the characteristic neurons or interneurons, especially in the hippocampal formation. This finding may help further elucidate the relationship or difference between gerbils and rats for brain function and behavioral pharmacology. Furthermore, our results suggest that cyclic-AMP and rolipram binding sites are predominantly distributed on the pyramidal cell layer of the hippocampal CA1 sector and that transient cerebral ischemia can cause marked reduction in these binding sites in the hippocampus.
...
PMID:Mapping of second messenger and rolipram receptors in mammalian brain. 132 28

Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.
...
PMID:Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain. 132 38

Foetal calf serum (FCS) and platelet-derived growth factor (PDGF)-stimulated incorporation of [3H]thymidine into pig aortic smooth muscle cell (ASMC) DNA was decreased by agents that either stimulated the synthesis (forskolin) or inhibited the breakdown (3-isobutyl-1-methylxanthine, IBMX) of cAMP. FCS-stimulated incorporation of [3H]thymidine into DNA was also reduced by selective inhibitors of cAMP-specific phosphodiesterase (PDE IV) (Ro-20-1724, rolipram) and cGMP-inhibited cAMP PDE (PDE III) (SK&F 94836). IBMX, Ro-20-1724, rolipram and SK&F 94836 enhanced forskolin inhibition of DNA synthesis. Alone, rolipram was a relatively weak inhibitor of FCS-induced ASMC DNA synthesis (IC25 greater than 20 microM); however, in the presence of a threshold concentration of SK&F 94836 (20 microM), the potency of rolipram increased (IC25 = 4 microM), suggesting synergy in the actions of PDE III and PDE IV inhibitors. SK&F 94836 and rolipram elicited 30% and 37%, respectively, reductions in FCS-induced ASMC proliferation and potentiated the inhibitory actions of forskolin. PDE III and PDE IV inhibitors alone, exerted minimal effects on ASMC cAMP levels after a short term (10 min) or long-term (2 or 24 hr) exposure, but enhanced forskolin-induced accumulation of cAMP. ASMC spontaneously released cAMP into the extracellular medium, a process that was increased by forskolin. PDE III and PDE IV inhibitors had no effect alone on cAMP extrusion but enhanced the effect of forskolin. Exposure of ASMC to forskolin or SK&F 94836 for 15 min increased the activity ratio (AR) of cAMP-dependent protein kinase from 0.05 to 0.17 and 0.23, respectively. Ro-20-1724, alone, did not affect cAMP-dependent protein kinase but enhanced the stimulatory effect of forskolin (AR = 0.37) and SK&F 94836 (AR = 0.27). Agents that increased cGMP synthesis (glycerol trinitrate, atrial natriuretic factor) or decreased its hydrolysis by selectively inhibiting cGMP-specific PDE (PDE V) (zaprinast) exerted no effects on FCS- or PDGF-stimulated [3H]thymidine incorporation into DNA either alone or in combination. The cytosolic fraction of pig ASMC contained four cyclic nucleotide PDEs which were categorized as PDE V, Ca2+/calmodulin-stimulated PDE (PDE I), PDE III and PDE IV. PDE I and III activities were also associated with the particulate fraction. The results demonstrate that inhibitors of PDEs III and IV alone or in combination with forskolin, reduce ASMC DNA synthesis and proliferation, through an action likely to involve elevation of intracellular cAMP. In contrast, inhibition of cGMP hydrolysing PDE subtypes (I and V) exerted no effect on DNA synthesis in this cell type.
...
PMID:Inhibition of pig aortic smooth muscle cell DNA synthesis by selective type III and type IV cyclic AMP phosphodiesterase inhibitors. 132 64


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---