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

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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)

Thrombin is thought to stimulate responsive cells by cleaving cell-surface receptors coupled to intracellular second-messenger-generating enzymes via G-proteins. In order to understand this process better, we have examined the regulation of adenylate cyclase by thrombin in the megakaryoblastic HEL cell line and compared it with platelets. A notable difference was found. In HEL-cell membrane preparations, thrombin inhibited cyclic AMP (cAMP) formation by a pertussis-toxin-sensitive mechanism comparable with that observed in platelets. In contrast, when added to intact HEL cells, thrombin activated adenylate cyclase and caused an increase in cAMP formation synergistic with that produced by forskolin and prostaglandin I2. This increase, which was not seen with platelets, was accompanied by an increase in cAMP metabolism by phosphodiesterase. Like other responses to thrombin, the increase in cAMP formation required proteolytically active thrombin and was subject to homologous desensitization. An equivalent response could be evoked by the addition of a polypeptide, derived from the N-terminus of the thrombin receptor, that has been shown to activate the receptor. The effects of thrombin could not, however, be reproduced by the addition of phorbol ester and the Ca2+ ionophore, A23187, nor be prevented with inhibitors of arachidonate metabolism. Preincubation of the cells with adrenaline, which inhibited Gs-mediated activation of adenylate cyclase, or pertussis toxin, which inhibited phospholipase C activation, had no effect on thrombin-induced cAMP formation. These results suggest that thrombin can regulate cAMP formation by two different mechanisms. First, thrombin can inhibit adenylate cyclase in a Gi-dependent manner. This effect predominates in HEL-cell membrane preparations, as it does in platelets, but is not detectable when thrombin is added to intact HEL cells. Instead, in intact HEL cells thrombin activates adenylate cyclase. Although clearly receptor-mediated, this response does not appear to involve Gi, Gs, protein kinase C, eicosanoid formation or changes in the cytosolic Ca2+ concentration.
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PMID:Dual regulation of cyclic AMP formation by thrombin in HEL cells, a leukaemic cell line with megakaryocytic properties. 131 10

Nucleotide pyrophosphatase (NPPase) activity is markedly elevated in cultured skin fibroblasts from patients of Lowe's syndrome. cDNA clones encoding the NPPase were isolated using synthetic oligonucleotide probes designed on the basis of partial amino acid sequence of the enzyme purified from human placenta. The complete sequences of these clones yielded a merged sequence of 3508 bases. The polypeptide chain of the enzyme was deduced to comprise 873 amino acids with a calculated molecular weight of 99,703 and had the characteristics of a class II transmembrane protein. Ten potential N-glycosylation sites were detected in the protein. RNA blot analysis showed that human fibroblasts contain two minor mRNAs of 7.0 and 8.2 kb, respectively, in addition to a major 3.6-kb species that coincides with the merged cDNA in size. A computer search of a nucleotide sequence data-base revealed that plasma cell membrane glycoprotein PC-1, whose function was unknown at the time, is identical with the NPPase. Comparison of the amino acid sequence of the NPPase with the active site sequence of bovine 5'-nucleotide phosphodiesterase allowed the assignment of a putative active site domain to the central region of the COOH-terminal extracellular domain of the NPPase. The gene for human NPPase was localized to chromosome 6 at q22-q23 by in situ hybridization with a fragment of the NPPase cDNA.
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PMID:Molecular cloning of cDNAs for human fibroblast nucleotide pyrophosphatase. 131 2

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.
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PMID:Insulin induced phosphorylation and activation of the cGMP-inhibited cAMP phosphodiesterase in human platelets. 132 13

Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues. To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains. Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes. One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J. K., Kadlecek, A., Sherbert, C. H., Seger, D., Sonnenburg, W. K., Charbonneau, H., Novack, J. P., and Beavo, J. A. (1992) J. Biol. Chem. 267, 18676-18682). This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone. The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE. Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin. Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line. These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes. Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides.
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PMID:A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs. Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase. 132 32

A phosphodiesterase from bull seminal plasma was purified to homogeneity. The purification procedure involved sequential column chromatographies on DEAE-Sephadex A-50, ConA-Agarose, chromatofocusing and AMP-Agarose. The final yield was about 20% with a 3000-fold purification. As indicated by chromatofocusing, the enzyme is an acidic protein (pI approximately 4.6) and owing to its interaction with Concanavalin A it is also a glycoprotein. The SDS-PAGE showed that the purified phosphodiesterase seemed to be constituted of a single polypeptide chain of about 125 kDa. The enzyme did not show an absolute substrate specificity. Thus, it was able to hydrolyze 4-nitrophenyl ester of 5'-TMP (but not of 3'-TMP), cAMP, nucleic acids as well as NAD+, ADP and ATP. According to its enzymatic properties, bull seminal plasma phosphodiesterase is to be considered an oligonucleate 5'-nucleotidohydrolase. In addition the seminal plasma phosphodiesterase also showed phosphonate esterase activity.
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PMID:Bull seminal plasma phosphodiesterase. Purification and general properties. 133 28

cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase-(PDE) were isolated from a human retinal library. The encoded polypeptide has 854 amino acid residues with calculated molecular mass of 98416 Da. Alignment of the deduced amino acid sequence with the previously analysed alpha-, beta- and alpha'-subunits of the bovine and mouse PDEs demonstrates highly significant similarities. We have also isolated, from a genomic library, two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene. The cloning of the human gene and the knowledge of its genomic organization will allow the rapid assessment of the role of this gene in the causation of human retinopathies.
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PMID:[Structural studies of cDNA and the gene for the beta-subunit of cGMP phosphodiesterase from human retina]. 133 85

The possibility that vasoactive intestinal polypeptide (VIP) may facilitate the nicotine-mediated induction of adrenal medullary tyrosine hydroxylase (TH) was investigated with primary cultures (5-7 days in vitro) of bovine adrenal chromaffin (BAC) cells. Exposure of BAC cells to 100 microM nicotine led to only a marginal increase in the amount of TH mRNA, TH protein, and TH activity. VIP, alone or in the presence of a phosphodiesterase inhibitor, produced a marked increase in TH mRNA, TH protein, and TH activity. Moreover, VIP together with nicotine, at concentrations that alone were devoid of effect, increased the amount of TH mRNA and TH activity. A synergistic effect of VIP and nicotine on cAMP accumulation in BAC cells was also apparent. The marginal effects of large doses of nicotine on both cAMP accumulation and TH induction were blocked completely by hexamethonium but were also partially inhibited by the VIP antagonist [p-chloro-D-Phe6,Leu17]-VIP. Nicotine may, therefore, stimulate the release of VIP from cultured BAC cells and VIP, in turn, by increasing cAMP, may synergize with nicotine to enhance TH gene expression.
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PMID:Vasoactive intestinal polypeptide facilitates tyrosine hydroxylase induction by cholinergic agonists in bovine adrenal chromaffin cells. 134 40

Peptide YY (PYY), found in intestinal endocrine cells, and neuropeptide Y (NPY), a structural analogue of PYY found in neurons, inhibit gastric, pancreatic, and intestinal fluid and electrolyte secretion. We examined the effects of these peptides on dispersed chief cells from guinea pig stomach. PYY and NPY, but not pancreatic polypeptide, starting at nanomolar concentrations, caused a 40-50% inhibition of secretin-, vasoactive intestinal polypeptide-, prostaglandin E2-, and forskolin-induced increases in chief cell adenosine 3',5'-cyclic monophosphate (cAMP) content and pepsinogen secretion. These inhibitory peptides did not alter pepsinogen secretion caused by cholecystokinin, carbamylcholine, A23187, 8-bromo-cAMP, or a phorbol ester. The inhibitory effects of PYY on chief cell cAMP production occurred within 30 s, were independent of phosphodiesterase activity, and did not affect the actions of cholera toxin. However, the inhibitory effects of PYY were abolished when chief cells were preincubated with pertussis toxin, an agent that uncouples inhibitory guanine nucleotide binding (G) proteins from their receptors. In gastric chief cells, PYY and NPY attenuate the stimulatory effects of secretagogues whose actions are mediated by changes in cellular levels of cAMP. PYY-induced attenuation of chief cell adenylate cyclase activity appears to involve activation of inhibitory G proteins.
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PMID:Actions of peptide YY and neuropeptide Y on chief cells from guinea pig stomach. 164 73

The cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is a peripheral enzyme activated in vivo by transducin. In vitro artificial activation can be achieved using trypsin. This was described as resulting from degradation of the inhibitory gamma subunit (2 copies/PDE molecule), leaving intact the alpha beta catalytic core. It was, however, observed that trypsin could induce the release of PDE (or solubilization) from the ROS membranes before its activation [Wensel, T. G. & Stryer, L. (1986) Proteins Struct. Funct. Genet. 1, 90-99]. Studying the time course of this solubilization, we were able to purify a trypsin-solubilized PDE still completely inhibited (i.e. with its two gamma subunits bound). The tryptic solubilization of PDE is therefore complete before any functional degradation of the gamma subunits occurs. It was recently suggested that this solubilization could coincide with the cleavage of a C-terminal fragment of the alpha subunit, which can be labeled by methylation of a terminal cysteine residue [Ong, O. C., Ota, I. M., Clarke, S. & Fung, B. K. K. (1989) Proc. Natl Acad. Sci. USA 86, 9238-9242]. We present the following evidence indicating that the C-terminus of the PDE beta subunit is mainly responsible for PDE anchorage to the ROS membrane. (a) The trypsin-solubilized PDE alpha beta gamma 2 has intact blocked N-termini. (b) It is still methylated on PDE alpha. (c) The C-terminus of PDE beta can also be labeled by methylation and its tryptic cleavage coincides well with the PDE solubilization. (d) Sequential cleavage of the alpha and beta polypeptides can also be detected by high-resolution gel electrophoresis: the first cleavage appears on the beta subunit and is completed when cleavage of the alpha subunit begins. The time course for cleavage of the gamma subunits appears to be slower than for the beta subunit and comparable to that of the alpha subunit. Upon longer trypsinization, a 70-kDa polypeptide appears which seems to be a degradation product of PDE beta. Gel-filtration analysis, however, shows that this 70-kDa fragment does not dissociate from the catalytic core.
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PMID:Activation and solubilization of the retinal cGMP-specific phosphodiesterase by limited proteolysis. Role of the C-terminal domain of the beta-subunit. 164 45

We have cloned a 4.2-kilobase pair (kb) cDNA that encodes the cyclic GMP-stimulated phosphodiesterase (cGS PDE) from a bovine adrenal cortex library. The 921-residue polypeptide deduced from the cDNA nucleotide sequence is nearly identical with the complete amino acid sequence of the cGS PDE purified from a soluble bovine heart extract. Moreover, PPD-S49 cells transfected with the cGS PDE cDNA express a soluble cAMP hydrolytic activity that is enhanced by cGMP. Total RNA isolated from several bovine tissues were screened for cGS PDE transcript by Northern blot analysis. The cGS PDE cDNA appears to hybridize to a single 4.5-4.6-kb mRNA species. Although the cGS PDE mRNA is most abundant in the adrenal cortex, it is also concentrated in the adrenal medulla and heart and in anatomically distinct regions of the brain and kidney. A mRNA species encoding a putative variant cGS PDE isoform was detected by RNase protection. Total RNA isolated from adrenal cortex, adrenal medulla, liver, kidney, trachea, lung, spleen, and T-lymphocytes completely protected a 452-base riboprobe encoding 100 residues of the adrenal cortex cGS PDE amino terminus. In contrast, RNAs isolated from brain (cerebral cortex, hippocampus, and basal ganglia) protected only 268 bases of this riboprobe. The RNase protection pattern of this same probe using heart RNA showed major bands at both 268 and 452 bases, suggesting that two different cGS PDE mRNA species are expressed. These results indicate that the cGS PDE is widely expressed in a variety of tissues. Moreover, these studies suggest that at least one different cGS PDE isoform having a structurally distinct amino-terminal domain is expressed in brain and heart.
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PMID:Molecular cloning of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase cDNA. Identification and distribution of isozyme variants. 165 33


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