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)

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.
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PMID:ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803. 776 63

Inhibition of a Zn(2+)-glycerophosphocholine cholinephosphodiesterase by thiols or tellurites were examined mechanistically. Inactivation of the phosphodiesterase by thio-carboxylates, which was due to the removal of Zn2+ in the catalytic site, was enhanced by introduction of an amino group in the structure of thiols, suggesting the presence of an anionic site adjacent to a Zn2+ site. In support of the suggestion, it was found that thiols, associable with both a Zn2+ site and an anionic site, were more potent reversible inhibitors; dimethylaminoethanethiol (Ki, 17 microM), diethylaminoethanethiol (Ki, 1.2 microM) and thiocholine (Ki, 2.6 microM). Meanwhile, the inhibition of the phosphodiesterase by tellurites is ascribed to the binding of tellurite anions to a Zn2+ site, based on the protective action of tellurite anions against the inactivation of the enzyme by EDTA. Moreover, the inhibition of the phosphodiesterase by tellurites was prevented by phosphate ions, which expressed the protective effect against EDTA inactivation. In further support, it was observed that tellurite and thiocholine appeared to interact with active site in an additive manner, in contrast to a synergistic action between tellurites and quaternary ammonium compounds such as acetylcholine or choline.
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PMID:Involvement of both a Zn2+ site and an anionic binding site in the selective inhibition of a Zn(2+)-glycerophosphocholine cholinephosphodiesterase by thiols and tellurites. 778 39

Addition of NH4+ to STreptomyces griseus 2682 cells grown in NO3- containing medium resulted in a rapid decline in glutamine synthetase activity due to covalent modification of the enzyme. The NH4+ promoted inactivation of the enzyme was inhibited by the ADP-ribosyltransferase inhibitor 3-methoxybenzamide. In the presence of ADP-ribosyltransferase activity the purified glutamine synthetase was also inhibited by NAD+ in a concentration-dependent manner. ADP-ribosylation of glutamine synthetase was demonstrated in vitro by showing the incorporation of labeled ADP-ribose from [alpha-32P]NAD+ into glutamine synthetase subunits. Beside ADP-ribosylation, adenylylation of glutamine synthetase was also shown in S. griseus since phosphodiesterase I treatment reactivated the enzyme in crude extracts of NH(4+)-shocked cells. Glutamine synthetase was also inhibited and modified by ATP in crude cellular extracts. These results suggest that in S. griseus 2682 ADP-ribosylation of glutamine synthetase could be an alternative modification to adenylylation to regulate glutamine synthetase activity.
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PMID:Modification of glutamine synthetase in Streptomyces griseus by ADP-ribosylation and adenylylation. 798 May 20

Exposure of endothelial cells (ECs) to thrombin or cytokines leads to major changes in their biochemical properties, which confer procoagulant activities. Stimulated ECs express the procoagulant glycoprotein tissue factor (TF). Although some TF is expressed on the apical surface of the cells, most is deposited as a cryptic pool in the subendothelial matrix. This matrix-associated TF may play a role in thromboembolic complications associated with alterations in the integrity of the EC monolayer. We have measured TF activity on the surface and in the subcellular matrix of human saphenous vein ECs in culture, by assaying the TF-dependent formation of activated factor X in the presence of factor VII. The subcellular matrix was prepared by exposure of ECs to ammonium hydroxide. Incubation of ECs for 4 h with 1 U/ml human thrombin induced TF expression on the apical cell surface and in the matrix. Activity in the matrix was 4.1 +/- 0.5 times greater than on the cell surface. Pentoxifylline inhibited the expression of TF both on the cell surface and in the matrix. The EC50 was on the order of 3.9 mM in both cases. No signs of cell toxicity were observed at this concentration of pentoxifylline. Similar effects were obtained with trequinsin (HL 725), a phosphodiesterase inhibitor, with an EC50 of 40 microM. This suggests that an increase in cAMP may be involved in the mechanism of action of pentoxifylline. Inhibition of TF deposition in the matrix may be important in the prevention of thromboembolic episodes in conditions where ECs either retract or are removed by major injury.
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PMID:Inhibitors of phosphodiesterase (pentoxifylline, trequinsin) inhibit apical and subcellular matrix expression of tissue factor in cultured human endothelial cells. 869 70

The development of targeted, bidentate photoaffinity reagents for mapping the interacting domains of calmodulin (CaM) with the enzymes that it regulates required the synthesis and evaluation of the binding affinity of various phenothiazines. These photoaffinity reagents would possess a photoactive 3-azidophenothiazine group for cross-linking the hydrophobic binding domain of CaM, a second photoactive benzophenone group that would be activated at a different wavelength than the 3-azidophenothiazine group, and a suitable radiolabel. Difficulties were encountered in identifying those structural features that would be compatible with the introduction of a benzophenone group, with the solubility of these benzophenone-substituted phenothiazines, and with the ability of these phenothiazines to inhibit the calmodulin-mediated activation of phosphodiesterase. Solutions to this problem involved the preparation of phenothiazines possessing a quaternary ammonium salt, a zwitterionic amino acid, or a carbohydrate moiety. The phenothiazines that possessed photoactive 3-azido and benzophenone groups and in which one of the piperazine nitrogens in the side chain was converted to a quaternary, N-methylammonium iodide inhibited the calmodulin-mediated activation of phosphodiesterase at a level comparable to that of chlorpromazine.
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PMID:Synthesis and binding affinity of bidentate phenothiazines with two different photoactive groups. 897 55

Properties of active site of Zn2+-glycerophosphocholine cholinephosphodiesterase from ox brain were examined using substrates and inhibitors of the phosphodiesterase. The anionic binding site expressed a selectivity for a positively-charged group. Meanwhile, the glyceryl moiety-binding site appeared to be a narrow crevice of a limited size, excluding the entry of acylglycerophospholipids containing long acyl chains. While endogenous quaternary ammonium compounds such as phosphocholine, choline or carnitine inhibited the enzyme, divalent metal ions such as Co2+, Mn2+ or Zn2+ enhanced the activity by 1.5 to 2-folds. The pH dependence for the inhibition by phosphocholine or the hydrolysis of substrate implies the involvement of a basic amino acid residue with a pK value of 9.6-9.7, probably lysine, in the binding of phosphoryl group. In further support, the lysine modifiers such as trinitrobenzene sulfonic acid or diethylpyrocarbonate expressed some inactivation. The pH-rate profile indicates that an amino acid residue with a pK value of 10.2, presumably tyrosine, may participate as a nucleophile in the catalysis. This might be further supported by the inactivation of the enzyme by tyrosine modifiers such as tetranitromethane or HOI-generating system. Separately, the phosphodiesterase was observed to be susceptible to the action of hydrogen peroxide or peroxynitrite-generating system. From these results, it is implied that the phosphodiesterase may be affected by endogenous sources.
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PMID:Active site of brain Zn2+-glycerophosphocholine cholinephosphodiesterase and regulation of enzyme activity. 970 95

1. We examined the effects of noradrenaline on steady-state intracellular pH (pHi) and the recovery of pHi from internal acid loads imposed by the NH4+ prepulse technique in hippocampal CA1 neurones acutely dissociated from adult rats. 2. Under nominally HCO3--free conditions, acid extrusion was accomplished by a Na+-dependent mechanism, probably the amiloride-insensitive variant of the Na+-H+ exchanger previously characterized in both fetal and adult rat hippocampal neurones. In the presence of external HCO3-, acid extrusion appeared to be supplemented by a Na+-dependent HCO3--Cl- exchanger, the activity of which was dependent upon the absolute level of pHi. 3. Noradrenaline evoked a concentration-dependent and sustained rise in steady-state pHi and increased rates of pHi recovery from imposed intracellular acid loads. The effects of noradrenaline were not dependent upon the presence of external HCO3- but were blocked by substituting external Na+ with N-methyl-D-glucamine, suggesting that noradrenaline acts to increase steady-state pHi by increasing the activity of the Na+-H+ exchanger. 4. The effects of noradrenaline on steady-state pHi and on rates of pHi recovery from imposed acid loads were mimicked by beta1- and beta2-, but not alpha-, adrenoceptor agonists. The beta-adrenoceptor antagonist propranolol blocked the ability of noradrenaline to increase both steady-state pHi and rates of pHi recovery from acid loads. 5. The effects of noradrenaline on steady-state pHi and on pHi recovery rates following acid loads were not dependent on changes in [Ca2+]i. However, the effects of noradrenaline were blocked by pre-treatment with the adenylate cyclase inhibitor 2',5'-dideoxyadenosine and the cAMP-dependent protein kinase inhibitors Rp-adenosine-3',5'-cyclic monophosphorothioate (sodium salt; Rp-cAMPS) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide (H-89). 6. Forskolin, an activator of endogenous adenylate cyclase, and 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, mimicked the ability of noradrenaline to increase both steady-state pHi and rates of pHi recovery from imposed acid loads, as did Sp-cAMPS, a selective activator of cAMP-dependent protein kinase. The effect of forskolin on steady-state pHi was blocked by pre-treatment with Rp-cAMPS whereas the effect of Sp-cAMPS was enhanced by pre-treatment with the protein phosphatase inhibitor, okadaic acid. 7. Noradrenaline also increased steady-state pHi and rates of pHi recovery from imposed acid loads in cultured postnatal rat hippocampal neurones. In this preparation, the effects of noradrenaline were occluded by 18-24 h pre-treatment with cholera toxin. 8. We conclude that noradrenaline increases the activity of the Na+-H+ exchanger in rat hippocampal neurones, probably by inducing an alkaline shift in the pHi dependence of the antiport, thereby raising steady-state pHi. The effects of noradrenaline are mediated by beta-adrenoceptors via a pathway which involves the alpha-subunit of the stimulatory G-protein Gs (Gsalpha), adenylate cyclase, cAMP and the subsequent activation of cAMP-dependent protein kinase which, in turn, may phosphorylate the exchange mechanism.
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PMID:Effects of noradrenaline on intracellular pH in acutely dissociated adult rat hippocampal CA1 neurones. 976 38

Biopharmacological evaluations of the protective effects of L-carnitine (a naturally occurring quaternary ammonium compound) against doxorubicin-induced metabolic damage were carried out in isolated cardiac myocytes and in isolated rat heart mitochondria. Perfusion of the heart with DOX (0.5 mM) caused a significant 70% inhibition of palmitate oxidation in cardiac myocytes, while L-carnitine (5 mM) perfusion caused stimulation which accounted for 37%. Perfusion of the heart with L-carnitine after 10-min perfusion with DOX (0.5 mM) caused 88% reversal of DOX-induced inhibition of palmitate oxidation in cardiac cells. In rat heart mitochondria, DOX has no effect on either palmitate oxidation or acyl-CoA synthetase activity, whereas Enoximone (c-AMP-dependent phosphodiesterase inhibitor), caused a significant inhibition of palmitate oxidation and acyl-CoA activity (40 and 27%, respectively). The oxidation of palmitoyl-CoA, an index of carnitine palmitoyltransferse reaction was significantly inhibited by DOX as a function of DOX concentration. Preincubation of mitochondria with L-carnitine caused reversal of DOX-induced inhibition of palmitoyl-CoA oxidation depending on the concentration of L-carnitine. Moreover, L-carnitine treatment did not interfere with the cytotoxic effect of doxorubicin against the growth of solid Ehrlich carcinoma. The findings of this study may suggest that inhibition of fatty acid oxidation in the heart is at least a part of doxorubicin cardiotoxicity and that L-carnitine can be used to prevent the doxorubcin-induced cardiac metabolic damage without interfering with its antitumour activities.
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PMID:Reversal of doxorubicin-induced cardiac metabolic damage by L-carnitine. 1020 59

The mechanism of adaptation of the acrylamide producing strain Rhodococcus rhodochrous M8 to changes in ammonium concentrations in the medium was studied. An increase in the content of ammonium in the medium changed the activity of glutamine synthetase (GS) (EC 6.3.1.2) and glutamine dehydrogenase (GD) (EC 1.4.1.4), the enzymes of ammonium assimilation, as well as the activities of enzymes responsible for nitrile utilization: nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4). This also caused inhibition of activation of GS induced by phosphodiesterase (EC 3.1.4.1). Increases in the activities of nitrile hydratase and amidase and resistance of these enzymes to ammonium were observed in mutant of R. rhodichrous resistant to phosphotricine, an inhibitor of GS. An important role of GS in the mechanism of adaptation is suggested.
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PMID:[Adaptation of acrylamide producer Rhodococcus rhodochrous M8 to change in ammonium concentration in medium]. 1075 79

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


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