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)

The glutamine synthetase (EC 6.3.1.2) from the N2-fixing bacterium Azotobacter vinelandii was purified to homogeneity by heat treatment, ammonium sulfate precipitation and ion-exchange chromatography. The following molecular parameters were determined: molecular weight 640 000, subunit molecular weight 53 000, partial specific volume 0.710 cm3/g, isoelectric point 4.6, amino acid composition. Most of the molecules are composed of 12 identical subunits but active oligomers of other degrees of polymerization, apparently aggregates with 8, 10 and 24 subunits, were also detected to a lesser extent. The enzymatic activity is regulated via adenylylation-deadenylylation cycles: liberation of AMP was detected upon treatment of the adenylylated form with phosphodiesterase along with a change in the catalytic properties. Adenylylation in vivo is specifically induced by high extracellular ammonia levels. The Km values for the Mg2+-dependent formation of glutamine were independent of the degree of adenylylation for glutamate and ATP, but varied for ammonia. Furthermore the catalytic activity is regulated by several nitrogenous feedback inhibitors. The degree of inhibition in some cases was dependent on the substrate concentrations: the sensitivity towards glycine, alanine and serine decreased with a decreasing ammonia level, while the sensitivity towards ADP or AMP increased with a decreasing ATP concentration. Part of the enzyme (about 30%) seems to be attached to the plasma membrane while the main fraction is found in the cytosol.
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PMID:The glutamine synthetase from Azotobacter vinelandii: purification, characterization, regulation and localization. 2 57

The possible relationship between phosphatidyl serine synthesis by base-exchange and nervous activity has been investigated in the rat caudate nucleus. The rate of incorporation of L-serine into the phosphatidyl serine of slices from caudate nucleus is not affected by dopamine nor is it affected by the addition to dopamine of a cyclic phosphodiesterase inhibitor which would increase the endogenous cyclic-AMP levels. However, imidazole, a phosphodiesterase activator, clearly stimulates by more than 100% the phosphatidyl serine synthesis in the slices. The activation is not due to interaction at the catalytic site(s) of the base-exchange system, since it is neither observed in homogenates of caudate nucleus nor in cerebral microsomes at various pH values.
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PMID:Activation of phosphatidylserine synthesis: a possible mechanism of regulation of the base-exchange enzymic system. 4 72

The acyl carrier protein of citrate lyase contains adenine, phosphate, sugar, cysteamine, beta-alanine and pantoic acid in a molar ratio of 1:2:2:1:1:1. Peptides containing these components in the same stoichiometric relationship were isolated after proteolytic digestion of acyl carrier protein. All components were linked together in a single prosthetic group. This was released from the peptide by mild alkaline hydrolysis. Under these conditions a phosphodiester bond is cleaved which links the prosthetic group to a serine residue of the peptide. Incubation of the prosthetic-group-containing peptide with phosphodiesterase I yielded 4'-phosphopantetheine and adenylic acid. The 5'-AMP was not free but was substituted by presumably an acidic sugar residue, which was released by mild acid hydrolysis yielding free 5'-AMP. It was concluded from these results that the prosthetic group of citrate lyase acyl carrier protein consists of a substituted isomeric dephospho-CoA. This is bound to the protein by the 5'-phosphate group of adenylic acid. The 4'-phosphopantetheine residue is bound by a phosphodiester linkage to the 2' or 3' position of ribose and the remaining hydroxyl group of ribose is substituted with presumably an acidic sugar residue. The structural similarities of this prothetic group and coenzyme A are discussed and related to the catalytic properties of citrate lyase.
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PMID:The prosthetic group of citrate-lyase acyl-carrier protein. 17 9

1. 3'-Guanylyl-ethanol, 3'-guanylyl-propanol, and 3'-guanylyl-alpha-glycerol were synthesized by ribonuclease N1 [EC 3.1.4.8] using guanosine 2',3'-cyclic phosphate as a phosphate donor and various alcohols as phosphate acceptors. The yields of these phosphodiesters were 15%, 13.5%, 38.2%, respectively, with respect to phosphate donor under the optimum conditions. No phosphodiester was synthesized when 2-propanol was used as a phosphate acceptor. Thus, primary alcoholic hydroxyl groups may be regarded as the preferred phosphate acceptor. 2. 3'-Guanylyl-glucose and 3'-guanylyl-ribose were synthesized using glucose and ribose as phosphate acceptors. Under the optimum conditions, the yields of guanylyl-glucose amounted to 52.0%, while that of guanylyl-ribose was much lower. The guanylyl-glucose can be regarded as 3'-guanylyl-6-glucopyranose, based on the results of periodate oxidation. 3. Neither hydroxyamino acids (serine and threonine) nor N-acetylserinamide could be phosphorylated under the conditions used for the above phosphorylations. 4. 3'-Guanylyl-glycerol obtained as above was hydrolyzed by snake venon phosphodiesterase to produce glycerol 3-phosphate. The latter consisted of L-glycerol 3-phosphate (ca 17%) and the D-isomer (ca. 83%). Ribonuclease N1 thus catalyzes an asymmetric synthesis.
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PMID:Synthesis of various phosphodiesters and phosphomonoesters with ribonuclease N. 18 80

A variety of 6- and 8-substituted analogs of cAMP (cyclic adenosine 3:5-monophosphate) have been tested for their ability to increase activity of tyrosine aminotransferase (EC 2.6.1.5) in cultured Reuber H35 hepatoma cells. Some analogs, particularly the 8-thio-substituted ones, produced effects approximately equivalent to those generated by N-6, O2'-dibutyryl cAMP. In contrast, cAMP and its O-2-monobutyryl derivative were relatively ineffective even at very high concentrations, whereas three other analogs actually depressed the activity of the aminotransferase. Changes in enzyme activity generated by the various analogs were paralleled closely by changes in the relative rate of aminotransferase synthesis. An excellent correlation was found to exist between the ability of any given analog to influence the activity of tyrosine aminotransferase and that of phosphoenolpyruvate carboxykinase (EC 4.1.1.32). A similar correlation was found to exist between the ability of various analogs to evelate the activity of these enzymes and to inhibit reversibly the growth of H35 cells. Only one of five inhibitors of cAMP phosphodiesterase activity tested produce any increase in aminotransferase activity when added alone. All of the 6- and 8-substituted analogs tested, including noniducers, stimulated f1 histone phosphorylation in crude rat liver extracts with approximately equal potencies. On the other hand, dibutyryl cAMP was only a weak activator of protein kinase in vitro, even though it is a potent enzyme inducer. A possible resolution of this apparent discrepancy has been provided by preliminary analyses of site-specific f1 histone phosphorylation in whole cells. Only compounds active as aminotransferase inducers are capable of stimulating phosphorylation of the serine-37 residue of endogenous f1 histone (3- to 10-fold).
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PMID:Effects of 6- and 8-substituted analogs of adenosine 3':5'-monophosphate on phosphoenolpyruvate carboxykinase and tyrosine aminotransferase in hepatoma cell cultures. 23 87

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.
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PMID:Phosphorylation by casein kinase II alters the biological activity of calmodulin. 131 63

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.
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PMID:Phosphorylation of the Epstein-Barr virus BZLF1 immediate-early gene product ZEBRA. 131 87

Some of the acute actions of insulin may be mediated by an enzyme-modulating inositol phosphate glycan, produced by the insulin-sensitive hydrolysis of glycosyl-phosphatidylinositol (GPI) that is structurally similar to a membrane protein anchor. An inositol glycan fragment from the structurally characterized Trypanosoma brucei variant surface glycoprotein GPI anchor is evaluated for insulin-mimetic antilipolytic activity. The fragment specifically and dose-dependently inhibits isoproterenol-stimulated lipolysis. Like the effect of insulin, glycan-induced antilipolysis is blocked by the low Km cAMP phosphodiesterase inhibitor imazodan (CI-914) and the serine/threonine phosphatase inhibitor, okadaic acid, suggesting that the activation of both cAMP phosphodiesterase and serine/threonine protein phosphatases are necessary. Moreover, this fragment causes a specific and dose-dependent inhibition of both microsomal glucose-6-phosphatase (EC 3.1.3.9) and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activity. Additionally, direct addition of the glycan to hepatocytes caused marked inhibition of glucose production from pyruvate. These results suggest that the direct modification of the activities of these two gluconeogenic enzymes by an inositol glycan may play a role in the inhibition of glucose output by insulin and provide the first evidence for the insulin-mimetic properties of a chemically characterized inositol glycan.
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PMID:An inositol phosphate glycan derived from a Trypanosoma brucei glycosyl-phosphatidylinositol mimics some of the metabolic actions of insulin. 132 96

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.
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PMID:Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain. 132 38

Parafusin, a cytosolic phosphoglycoprotein of M(r) 63,000, is dephosphorylated and rephosphorylated rapidly in a Ca(2+)-dependent manner upon stimulation of exocytosis in vivo in wild-type (wt) Paramecium. In contrast, the temperature-sensitive exocytosis mutant nd9, grown at the nonpermissive temperature (27 degrees C), does not exocytose or dephosphorylate parafusin upon stimulation in the presence of Ca2+; grown at the permissive temperature (18 degrees C), nd9 cells show a wt phenotype. Parafusin contains two types of phosphorylation sites: one where glucose 1-phosphate is added by an alpha-glucose-1-phosphate phosphotransferase and removed by a phosphodiesterase and one where phosphate from ATP is added directly to a serine residue by a protein kinase and removed by a phosphatase. We show here that, in cell fractions from wt Paramecium, both reactions can be carried out in vitro by using uridine(5'-[beta-[35S]thio])diphospho(1)-glucose (UDP[beta 35S]-Glc) and [gamma-32P]ATP, respectively. The characteristics of these pathways are different. Specifically, in the presence of Ca2+, the amount of UDP[beta 35S]-Glc label in parafusin is reduced. In contrast, identical labeling experiments with [gamma-32P]ATP show that Ca2+ enhances labeling of parafusin. Mg2+ had no appreciable effect on either labeling. Removal of the UDP[beta 35S]-Glc label on parafusin in the presence of Ca2+ correlates with the in vivo dephosphorylation seen upon exocytosis. Incubations with UDP[beta 35S]-Glc were then performed with homogenates and nd9 cell fractions grown at 27 degrees C under the ionic conditions used for wt cells. These labelings were not affected by Ca2+, in contrast to results from wt cells but in accord with those obtained earlier with nd9-27 mutant cells in vivo. Factors responsible for both dephosphorylation and Ca2+ sensitivity were found in the high-speed pellet (P2) in wt cells, suggesting that the putative phosphodiesterase is in this fraction and that the defect in the mutant nd9-27 residues in the Ca2+ activation of the phosphodiesterase. We conclude that the in vivo dephosphorylation of parafusin that occurs upon exocytosis is a dephosphoglucosylation due to removal of the alpha-glucose 1-phosphate and more generally that carbohydrates on cytoplasmic glycoproteins may be cyclically added and/or removed in response to extracellular stimuli.
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PMID:Carbohydrate cycling in signal transduction: parafusin, a phosphoglycoprotein and possible Ca(2+)-dependent transducer molecule in exocytosis in Paramecium. 133 6


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