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)

A new purification procedure for the isolation of the "unlinking" enzyme, which hydrolyzes the phosphodiester bond between 5;-terminal uridylic acid of the encephalomyocarditis viral RNA and protein VPg has been developed. The enzyme (tyrosine-(5;P-->O)-uridylylpolynucleotide phosphodiesterase, Y-pUpN PDE) was purified from frozen mouse carcinoma Krebs II cells. The purification procedure included ammonium sulfate fractionation of the cell extract, pH fractionation by acidification of the protein solution to pH 4.0, cation-exchange chromatography on CM-52-cellulose, chromatofocusing, and size-exclusion HPLC on a TSK 2000 SW column. The enzyme was shown to exist as several forms characterized by different isoelectric points (ranging from 4.0 to 5. 2) and molecular masses. The pH fractionation and ion-exchange chromatography on CM-cellulose influenced the pI and molecular mass values for each form (pI increased, whereas molecular mass decreased from 30 to 26 kD). The employment of these two stages removed (almost completely) an accompanying proteolytic activity, which co-purified with Y-pUpN PDE and digested free VPg. The molecular mass of 26 kD determined by HPLC for the native form coincided with the molecular mass of the major protein band determined by SDS-PAGE for the denatured form of the enzyme.
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PMID:Isolation from ascites carcinoma Krebs II cells of an unlinking enzyme hydrolyzing a covalent bond between picornavirus RNA and VPg. 1109 68

The gene encoding periplasmic 2',3'-cyclic phosphodiesterase in Yersinia enterocolitica O:8 (designated cpdB), was cloned and expressed in Escherichia coli. This enzyme enables Y. enterocolitica to grow on 2',3'-cAMP as a sole source of carbon and energy. Sequencing and analysis of a 3 kb ECO:RI fragment containing the cpdB gene revealed an open reading frame of 1179 bp, corresponding to a protein with a molecular mass of 71 kDa. The first 25 amino acid residues show features of a typical prokaryotic signal sequence. The predicted molecular mass of the mature peptide is therefore in agreement with the molecular mass estimated by SDS gel electrophoresis (68 kDa). The putative cpdB promoter region contains two possible -10 and -35 regions. Furthermore, the 5' untranslated region contains sequences with significant homology to the cyclic AMP-cyclic AMP receptor protein binding site and the sigma(28) consensus. This region is interrupted by an enterobacterial repetitive intergenic consensus (ERIC) sequence. Deletion of the ERIC element from the cpdB promoter region had no effect on cpdB expression. In the 3' untranslated region, a possible rho-independent transcriptional terminator was identified. The deduced amino acid sequence of the Y. enterocolitica CpdB protein shows 76% identity with CpdB of Salmonella typhimurium and E. coli. CpdB of Y. enterocolitica is exported to the periplasmic space. An isogenic Y. enterocolitica cpdB mutant strain, constructed by allelic exchange, was no longer able to grow on 2',3'-cAMP as sole source of carbon and energy. The CpdB mutant showed no significant change in virulence in an oral and intravenous mouse infection model.
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PMID:Cloning and characterization of the gene encoding periplasmic 2',3'-cyclic phosphodiesterase of Yersinia enterocolitica O:8. 1116 Aug 14

Two isoforms of ADPglucose pyrophosphatase/phosphodiesterase (AGPPase) have been characterized using barley leaves (Hordeum vulgare L.). Whilst one of the isoforms, designated as soluble AGPPase1 (SAGPPase1), is soluble in low ionic strength buffers, the other, SAGPPase2, is extractable using cell wall hydrolytic enzymes or high salt concentration solutions, thus indicating that it is adventitiously bound to the cell wall. Both AGPPase isoforms are highly resistant to SDS, this characteristic being utilized to purify them to homogeneity after zymographic detection of AGPPase activity in SDS-containing gels. N-terminal and internal amino acid sequencing analyses revealed that both SAGPPase1 and SAGPPase2 are distinct oligomers of the previously designated HvGLP1, which is a member of the ubiquitously distributed group of proteins of unknown function designated as germin-like proteins (GLPs).
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PMID:Two isoforms of a nucleotide-sugar pyrophosphatase/phosphodiesterase from barley leaves (Hordeum vulgare L.) are distinct oligomers of HvGLP1, a germin-like protein. 1117 8

A phosphodiesterase was purified from the venom of the snake Bothrops alternatus by a combination of gel filtration and ion exchange chromatographies. In SDS-PAGE, the enzyme gave a single band with a molecular mass of 105 kDa, which was unaltered in the presence of beta-mercaptoethanol, indicating that the protein contained no subunits. A single protein band was also observed in native PAGE. There were no contaminating 5'-nucleotidase, alkaline phosphatase and protease activities. The enzyme was recognized by commercial bothropic antiserum and gave a single band in immunoblotting. The enzyme had a pH optimum in the range of 7.5-9.5 and the optimum temperature was 60 degrees C, with activity being rapidly lost within 1 min at > or = 70 degrees C. The Km of the enzyme was 2.69 mM. PDE activity was potentiated by cobalt and, to a lesser extent, by calcium, whereas copper, manganese, zinc, EDTA, and beta-mercaptoethanol were inhibitory. These properties show that this enzyme is very similar to that isolated from other snake venoms.
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PMID:Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom. 1263 51

Orthovanadate (vanadate) as well as insulin stimulated phosphodiesterase 3 (PDE3) in the particulate fraction of rat hepatocytes. The vanadate-induced activations of PDE3 and mitogen-activated protein kinase (MAPK) were inhibited by H-89 and PD98059, suggesting that the MAPK activation via cAMP-dependent protein kinase (PKA) and MAPK kinase is involved in the vanadate action. On the other hand, the insulin-induced activations of PDE3 and Akt were inhibited by wortmannin, suggesting involvement of the Akt activation via phosphatidylinositol 3-kinase (PI3K) in the insulin action. The vanadate-induced activations of PKA and PDE3 were inhibited in part by propranolol or genistein, suggesting that vanadate may exert its actions via dual signaling pathways of beta-adrenergic receptors and receptor tyrosine kinases of growth factors. Vanadate, in contrast to insulin, did not promote the phosphorylation of insulin receptor substrate-1. The vanadate-induced increase in the phosphorylation of a main isoform of MAPKs, p44 protein, was detected by immunoblotting migration patterns of SDS-PAGE. A partially purified PDE3 activity was increased by addition of MAPK or Akt to the reaction mixture, suggesting that MAPK as well as Akt acts upstream of PDE3. The activation of PDE3 by insulin was independent of a transient increase in the MAPK activity, probably due to the dephosphorylated inactivation mediated by the induced activation of MAPK phosphatases (MKPs). Vanadate did not affect the MKP activity. These results indicate that vanadate stimulates the particulate PDE3 activity by activating mainly p44 MAPK via a PKA-dependent process, and that it differs from insulin with regard to a phosphorylation cascade of PDE3 activation.
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PMID:Orthovanadate stimulates cAMP phosphodiesterase 3 activity in isolated rat hepatocytes through mitogen-activated protein kinase activation dependent on cAMP-dependent protein kinase. 1518 19

Small membranous vesicles, between 25- and 75-nm diameter, were collected by high-speed centrifugation from the ram cauda epididymal fluid and were found to be normal constituents of this fluid and of the seminal plasma. The SDS-PAGE protein pattern of these vesicles was specific and very different from that of the caudal fluid, seminal plasma, sperm extract, and cytoplasmic droplets. After two-dimensional electrophoresis separation and mass spectrometry analysis, several proteins were identified and grouped into i) membrane-linked enzymes, such as dipeptidyl peptidase IV (DPP-IV), neprilysin (NEP), phosphodiesterase-I (E-NPP3), and protein G-beta; ii) vesicle-associated proteins, such as lactadherin (MFEG8-PAS6/7) and vacuolar ATPase; iii) several cytoskeleton-associated proteins, such as actin, ezrin and annexin; and iv) metabolic enzymes. The presence of some of these proteins as well as several different hydrophobic proteins secreted by the epididymis was further confirmed by immunoblotting. These markers showed that the majority of the vesicles originated from the cauda epididymal region. The physical and biochemical characteristics of these vesicles suggest they are the equivalent of the exosomes secreted by several cell types and epithelium. The main membrane-linked proteins of the vesicles were not retrieved in the extract from cauda or ejaculated sperm, suggesting that these vesicles did not fuse with sperm in vivo.
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PMID:Identification, proteomic profiling, and origin of ram epididymal fluid exosome-like vesicles. 1563 28

Several extracellular DNases were detected after cultivation of Streptomyces aureofaciens B96 under submerged conditions. These DNases are nutritionally regulated and high content of amino acid nitrogen in cultivation medium repress their production. By varying cultivation conditions, there remained only two extracellular nuclease activities. The major one, extracellular endodeoxyribonuclease SaD I, was purified to homogeneity by ammonium sulfate precipitation, adsorption on Spheron, chromatography on Superose-12P followed by FPLC on MonoQ and final purification on HiTrapQ. The molecular weight of the purified SaD I determined by SDS-PAGE was 31 kDa. The DNase hydrolyses endonucleolytically both double-stranded and single-stranded circular and linear DNA. It does not cleave RNA and does not exhibit phosphodiesterase nor phosphomonoesterase activity. It requires a divalent cation (Zn2+, Co2+, Mn2+, Mg2+) and its activity optimum is at neutral pH (pH 7.2). The optimal temperature for DNA cleavage was 40 degrees C. Activity was strongly inhibited in the presence of phosphate, Hg2+, chelating agents or iodoacetate, but it was stimulated by addition of dimethyl sulphoxide.
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PMID:An extracellular endodeoxyribonuclease from Streptomyces aureofaciens. 1565 86

Cyclic nucleotide PDE4 (phosphodiesterase 4) inhibitors are being developed as potent anti-inflammatory drugs for use in chronic lung diseases, but the complexity of the PDE4 family has hampered this process. The four genes comprising the PDE4 family, PDE4A, PDE4B, PDE4C and PDE4D, are all expressed as multiple splice variants. The most widely used criterion to identify PDE4 variants expressed endogenously is their migration on SDS/PAGE. However, when a PDE4D3-selective antibody was used for immunoprecipitation, the pattern of expression obtained did not confirm the expression predicted by SDS/PAGE. This observation, together with the recent discovery of additional PDE4D transcripts, prompted us to re-evaluate the pattern of expression of these variants. The nine rat PDE4D splice variants, PDE4D1 to PDE4D9, were cloned, their electrophoretic properties compared, and their in vivo mRNA and protein levels determined. Using this approach, we found that the pattern of distribution of the PDE4D splicing variants is more complex than previously reported. Multiple variants co-migrate in single immunoreactive bands, and variant-selective antibodies were necessary to discriminate between splice variants. Tissues that were thought to express only PDE4D3, express three closely related proteins, with PDE4D8 and PDE4D9 as the predominantly expressed forms. In addition, activation of cAMP signalling produces phosphorylation and activation of variants other than PDE4D3, and expression of PDE4D mRNA does not always correlate with the pattern of protein expression. As PDE4 inhibitors have different affinities for distinct PDE4D splicing variants, our results indicate that a better definition of the pattern of PDE4 expression is required for target validation.
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PMID:Splice variants of the cyclic nucleotide phosphodiesterase PDE4D are differentially expressed and regulated in rat tissue. 1571 66

A role for the cAMP-dependent pathway in regulation of the cell wall in the model yeast Saccharomyces cerevisiae has recently been demonstrated. In this study we report the results of a phenotypic analysis of a Candida albicans mutant, characterized by a constitutive activation of the cAMP pathway due to deletion of PDE2, the gene encoding the high cAMP-affinity phosphodiesterase. Unlike wild-type strains, this mutant has an increased sensitivity to cell wall and membrane perturbing agents such as SDS and CFW, and antifungals such as amphotericin B and flucytosine. Moreover, the mutant is characterized by an altered sensitivity and a significantly reduced tolerance to fluconazole. The mutant's membrane has around 30% higher ergosterol content and the cell wall glucan was 22% lower than in the wild-type. These cell wall and membrane changes are manifested by a considerable reduction in the thickness of the cell wall, which in the mutant is on average 60-65 nm, compared to 80-85 nm in the wild-type strains as revealed by electron microscopy. These results suggest that constitutive activation of the cAMP pathway affects cell wall and membrane structure, and biosynthesis, not only in the model yeast S. cerevisiae but also in the human fungal pathogen C. albicans.
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PMID:Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans. 1578 49

Paired Y-organs secrete ecdysteroid hormones that control cycles of growth and molting in crustaceans. Y-Organs are regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide produced and released by the X-organ/sinus gland complex of the eyestalks. In the present studies, crab (Callinectes sapidus) Y-organs were incubated in vitro in the presence of [(35)S]methionine, and cyclic nucleotide analogs or experimental agents that influence the cAMP signaling pathway. In 4-hr incubations, 8-Br-cAMP and db-cAMP (but not 8-Br-cGMP) suppressed incorporation of [(35)S]methionine into Y-organ proteins; the effect of 8-Br-cAMP was concentration-dependent. Autoradiograms of radiolabeled Y-organ proteins separated on SDS-PAGE gels indicated the effect of 8-Br-cAMP was general (as opposed to selective) suppression of protein synthesis. Addition of both forskolin (an adenylyl cyclase activator) and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) likewise suppressed incorporation of [(35)S]methionine into Y-organ proteins. Cycloheximide (a protein synthesis inhibitor) suppressed incorporation of [(35)S]methionine into Y-organ proteins and secretion of ecdysteroids. The combined results suggest that cAMP is involved in regulation of protein synthesis in C. sapidus Y-organs. We are currently investigating the link of protein synthesis to ecdysteroid production, and the possibility of cross-talk between cAMP and other cellular signaling pathways in Y-organs.
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PMID:Regulation of protein synthesis in Y-organs of the blue crab (Callinectes sapidus): involvement of cyclic AMP. 1649 43


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