Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The glutathione transferases (GSTs) mediate the detoxification of a broad spectrum of electrophilic chemicals. We report here the identification and characterisation of a novel naturally occurring transition that changes codon 169 from GGC (Gly) to GAC (Asp) in the human Pi class GST, GSTP1. Expression of the variant in human HepG2 cells led to a small increase in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation compared to the wild-type protein. Asp(169) GSTP1-1 expressed at high levels in Escherichia coli displayed a small but significant increase in specific activity towards CDNB compared to Gly(169) GSTP1-1. The catalytic efficiency with CDNB was higher for Asp(169) GSTP1-1 compared to the wild-type enzyme, although the kinetic constants of the mutant and the wild-type enzyme towards glutathione were not different. Modelling indicated that the mutation does not appear to change protein conformation. The distribution of the genotypes in a normal healthy population (217 individuals) was 94.3% for the Gly/Gly genotype and 5.7% for the Gly/Asp genotype; no Asp/Asp genotypes were detected in this population. The frequency of the Asp(169) allele in the only oxidative stress-linked pathology that we have studied to date, i.e. alcoholic liver disease, was not significantly different from healthy controls. In conclusion, we have detected and characterised a novel SNP in GSTP1 that may play a role in modulating the activity of GSTP1-1.
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PMID:Detection and biochemical characterisation of a novel polymorphism in the human GSTP1 gene. 1756 37

Polychlorinated biphenyls (PCBs) are ubiquitious lipophilic environmental pollutants. Some of the PCB congeners and mixtures of congeners have tumor promoting activity in rat liver. The mechanism of their activity is not fully understood and is likely to be multifactorial. The aim of this study was to investigate if the resident liver macrophages, Kupffer cells, are important in the promoting activity of PCBs. The hypothesis of this study was that the inhibition of Kupffer cell activity would inhibit hepatic tumor promotion by PCBs in rats. To test our hypothesis, we studied the effects of Kupffer cell inhibition by dietary glycine (an inhibitor of Kupffer cell secretory activity) in a rat two-stage hepatocarcinogenesis model using 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153, a non-dioxin-like PCB) or 3,3',4,4'-tetrachlorobiphenyl (PCB-77, a dioxin-like PCB) as promoters. Diethylnitrosamine (DEN, 150 mg/kg) was administered to female Sprague-Dawley rats, which were then placed on an unrefined diet containing 5% glycine (or casein as nitrogen control) starting two weeks after DEN administration. On the third day after starting the diets, rats received PCB-77 (300 micromol/kg), PCB-153 (300 micromol/kg), or corn oil by i.p. injection. The rats received a total of 4 PCB injections, administered every 14 days. The rats were euthanized on the 10th day after the last PCB injection, and the formation of altered hepatic foci expressing placental glutathione S-transferase (PGST) and the rate of DNA synthesis in these foci and in the normal liver tissue were determined. Glycine did not significantly affect foci number or volume. PCB-153 did not significantly increase the focal volume, but increased the number of foci per liver, but only in the rats not fed glycine; PCB-77 increased both the foci number and their volume in both glycine-fed and control rats. Glycine did not alter the PCB content of the liver, but did increase the activity of 7-benzyloxyresorufin O-dealkylase (BROD) in liver microsomes from PCB-153 treated rats. However, glycine did not affect the induction of ethoxyresorufin O-dealkylase activity by PCB-77 in liver microsomes. Glycine diminished hepatocyte proliferation in PGST-positive foci, but not in normal tissue. Overall these results do not support the hypothesis that dietary glycine inhibits the promoting activities of PCBs. The observations that PCB-153 increased the number of foci per liver in control rats but not glycine-fed rats and that dietary glycine reduced cell proliferation in PGST-positive foci, however, do not allow us to completely rule out a role for dietary glycine. But the data overall indicate that Kupffer cells likely do not contribute to the tumor promoting activities of PCB-77 and PCB-153.
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PMID:The effect of dietary glycine on the hepatic tumor promoting activity of polychlorinated biphenyls (PCBs) in rats. 1770 65

The role of Ser-308 of murine D-aspartate oxidase (mDASPO), particularly its side chain hydroxyl group, was investigated through the use of site-specific mutational analysis of Ser-308. Recombinant mDASPO carrying a substitution of Gly, Ala, or Tyr for Ser-308 was generated, and fused to either His (His-mDASPO), or glutathione S-transferase, His, and S (GHS-mDASPO) at its N-terminus. Wild-type His-mDASPO or GHS-mDASPO or their mutant derivatives were expressed in Escherichia coli and purified by affinity chromatography. All purified recombinant proteins had functional DASPO activity. The Gly-308 and Ala-308 mutants had significantly higher catalytic efficiency towards D-Asp and N-methyl-D-Asp, and a higher affinity for flavin adenine dinucleotide (FAD) compared to the wild-type enzyme. The Tyr-308 mutant had lower catalytic efficiency and binding capacity. These results suggest that the side chain hydroxyl group of a critical residue of mDASPO, Ser-308, down-regulates enzymatic activity, substrate binding, and FAD binding. This study provides information on the active site of DASPO that will considerably enhance our understanding of the biological significance of this enzyme.
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PMID:Hyperactive mutants of mouse D-aspartate oxidase: mutagenesis of the active site residue serine 308. 1823 94

Glutathione transferases (GSTs) from the tau class (GSTU) are unique to plants and have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. A fluorodifen-induced GST isoezyme (GmGSTU4-4) belonging to the tau class was purified from Glycine max by affinity chromatography. This isoenzyme was cloned and expressed in Escherichia coli, and its structural and catalytic properties were investigated. The structure of GmGSTU4-4 was determined at 1.75 A resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). The enzyme adopts the canonical GST fold but with a number of functionally important differences. Compared with other plant GSTs, the three-dimensional structure of GmGSTU4-4 primarily shows structural differences in the hydrophobic substrate binding site, the linker segment and the C-terminal region. The X-ray structure identifies key amino acid residues in the hydrophobic binding site (H-site) and provides insights into the substrate specificity and catalytic mechanism of the enzyme. The isoenzyme was highly active in conjugating the diphenylether herbicide fluorodifen. A possible reaction pathway involving the conjugation of glutathione with fluorodifen is described based on site-directed mutagenesis and molecular modeling studies. A serine residue (Ser13) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione and enhance its nucleophilicity. Tyr107 and Arg111 present in the active site are important structural moieties that modulate the catalytic efficiency and specificity of the enzyme, and participate in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. A hitherto undescribed ligand-binding site (L-site) located in a surface pocket of the enzyme was also found. This site is formed by conserved residues, suggesting it may have an important functional role in the transfer and delivery of bound ligands, presumably to specific protein receptors.
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PMID:Crystallographic and functional characterization of the fluorodifen-inducible glutathione transferase from Glycine max reveals an active site topography suited for diphenylether herbicides and a novel L-site. 1901 49

Actin, the major component of the cytoplasmic skeleton, has been shown to exist in the nucleus. Nuclear actin functions in several steps of the transcription process, including chromatin remodelling and transcription initiation and elongation. However, as a part of PICs (pre-initiation complexes), the role of actin remains to be elucidated. In the present study, we identified RHA (RNA helicase A) as an actin-interacting protein in PICs. Using immunoprecipitation and immunofluorescence techniques, we have shown that RHA associates with beta-actin in the nucleus. A GST (glutathione transferase) pulldown assay using different deletion mutants revealed that the RGG (Arg-Gly-Gly) region of RHA was responsible for the interaction with beta-actin, and this dominant-negative mutant reduced the recruitment of Pol II (RNA polymerase II) into PICs. Moreover, overexpression or depletion of RHA could influence the interaction of Pol II with beta-actin and beta-actin-involved gene transcription regulation. These results suggest that RHA acts as a bridging factor linking nuclear beta-actin with Pol II.
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PMID:RNA helicase A acts as a bridging factor linking nuclear beta-actin with RNA polymerase II. 1930 9

The cDNA of a cystatin analogue was isolated from the spleen Smart cDNA library of large yellow croaker Pseudosciaena crocea (Lyccys). The open reading frame (ORF) of 354 nucleotides (nt) of Lyccys encodes a protein of 118 amino acids (aa), containing a 21-aa signal peptide and a 97-aa mature polypeptide. The deduced Lyccys possessed structural features of the Family II cystatins, including three evolutionally conserved motifs known to interact with the active sites of cysteine peptidases: Gly at the N-terminus (Gly(25)), Gln-X-Val-X-Gly motif (Q(69)LVAG(73)) and Pro-Try pair at the C-terminus (P(106)W(107)). Genomic analysis revealed that Lyccys gene, spanning 2297 nt, consisted of three exons and two introns. The Lyccys gene was constitutively expressed in all eight tissues examined although at different levels. Real-time PCR analysis revealed that Lyccys transcript in spleen and kidney was obviously up-regulated by poly(I:C) or inactivated trivalent bacterial vaccine, while in blood its expression was down-regulated. Immuno-electron microscopy showed that Lyccys was mainly localized to the rough endoplasmic reticulum (rER) or in the vesicular structures in spleen and kidney cells. Recombinant Lyccys protein fused with glutathione S-transferase (rLyccys) was shown to have remarkable protease-inhibitory activity and well affinity binding to papain (with a K(i) of 1.3x10(-13) M). An in vivo administration of rLyccys could significantly up-regulate the expression levels of large yellow croaker tumor necrosis factor-alpha2 (TNF-alpha2) and interleukin-10 in spleen and kidney, but to a lesser extent increase TNF-alpha1 expression. These results suggest that the Lyccys is a secreted inhibitor of cysteine proteinases, which may have an immunomodulatory function in inflammation response.
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PMID:Molecular and functional characterization of a cystatin analogue in large yellow croaker (Pseudosciaena crocea). 1932 54

Cytosolic GSTs (glutathione transferases) are a multifunctional group of enzymes widely distributed in Nature and involved in cellular detoxification processes. The three-dimensional structure of GmGSTU4-4 (Glycine max GST Tau 4-4) complexed with GSH was determined by the molecular replacement method at 2.7 A (1 A=0.1 nm) resolution. The bound GSH is located in a region formed by the beginning of alpha-helices H1, H2 and H3 in the N-terminal domain of the enzyme. Significant differences in the G-site (GSH-binding site) as compared with the structure determined in complex with Nb-GSH [S-(p-nitrobenzyl)-glutathione] were found. These differences were identified in the hydrogen-bonding and electrostatic interaction pattern and, consequently, GSH was found bound in two different conformations. In one subunit, the enzyme forms a complex with the ionized form of GSH, whereas in the other subunit it can form a complex with the non-ionized form. However, only the ionized form of GSH may form a productive and catalytically competent complex. Furthermore, a comparison of the GSH-bound structure with the Nb-GSH-bound structure shows a significant movement of the upper part of alpha-helix H4 and the C-terminal. This indicates an intrasubunit modulation between the G-site and the H-site (electrophile-binding site), suggesting that the enzyme recognizes the xenobiotic substrates by an induced-fit mechanism. The reorganization of Arg111 and Tyr107 upon xenobiotic substrate binding appears to govern the intrasubunit structural communication between the G- and H-site and the binding of GSH. The structural observations were further verified by steady-state kinetic analysis and site-directed mutagenesis studies.
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PMID:Crystal structure of Glycine max glutathione transferase in complex with glutathione: investigation of the mechanism operating by the Tau class glutathione transferases. 1953 82

In this paper, we report the molecular cloning of a novel stefin analogue from the spleen of large yellow croaker Pseudosciaena crocea (Lycstefin). The open reading frame (ORF) of 297 nucleotides (nt) of Lycstefin encodes a protein of 99 amino acids (aa) with a putative molecular weight of 11kDa, in which no signal peptide and potential N-glycoslation site are predicted. The deduced Lycstefin possesses the structural features of the mammalian stefins, including two conserved motifs known to interact with the active sites of family C1 cysteine peptidases: one glycine in the N-terminal region (G(6)) and Gln-Xaa-Val-Xaa-Gly motif (Q(48)LVAG(52)). It shares 32-47.5% aa sequence identity to the sequences found in mammals and other fish species and is rich in cysteine residues (seven cysteines). Genomic analysis revealed that Lyccys gene, 757 nt long, consisted of three exons and two introns. The Lycstefin gene was constitutively expressed in various tissues examined although at different levels. Upon stimulation with poly(I:C) or inactivated trivalent bacterial vaccine, Lycstefin transcript was significantly up-regulated in spleen and head kidney while down-regulated in blood. Immuno-electron microscopy showed that Lycstefin was mainly localized in the cytoplasm of spleen cells of large yellow croaker, and also in the nucleus. Recombinant Lycstefin protein fused with glutathione S-transferase (rLycstefin) was shown to have strong inhibitory activity against papain with a K(i) of 1.3x10(-13)M. The in vivo experiments revealed that Lycstefin could not modulate the expression levels of large yellow croaker tumor necrosis factor-alpha2 (TNF-alpha2) and interleukin-10 in spleen and head kidney. To our knowledge, this is the first report on the molecular and functional identification of a stefin analogue in bony fish.
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PMID:Molecular and functional characterization of a novel stefin analogue in large yellow croaker (Pseudosciaena crocea). 1965 Nov 53

Glycine soja is a species of soybean that survives in adverse environments including high salt and drought conditions. We constructed a cDNA library from G. soja seedlings treated with NaCl and isolated a glutathione S-transferase gene (GsGST: GQ265911) from the library. The cDNA encoding GsGST contains an open reading frame of 660 bp and the predicted protein belongs to the tau class of GST family proteins. Tobacco plants over-expressing the GsGST gene showed sixfold higher GST activity than wild-type plants. Transgenic tobacco plants exhibited enhanced dehydration tolerance. T(2) transgenic tobacco plants showed higher tolerance at the seedling stage than wild-type plants to salt and mannitol as demonstrated by longer root length and less growth retardation.
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PMID:Over-expression of a glutathione S-transferase gene, GsGST, from wild soybean (Glycine soja) enhances drought and salt tolerance in transgenic tobacco. 2038 60

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes catalyzing the conjugation of a broad range of toxicologically important halogenated compounds to the tripeptide glutathione (GSH) with concomitant halogen ion release. In the present work, a rapid quantitative screening method for GSTs based on colorimetric measurement of halogen ions released from halogenated xenobiotics was developed. The assay is based on the color formation resulting from the reaction of Hg(SCN)(2) with the released halogen ion of the substrate in the presence of Fe(3+). The color intensity is proportional to the extent of the catalytic reaction, allowing a quantitative measurement of the GST catalytic activity. The assay can be performed using purified recombinant enzyme (the isoenzyme GmGSTU4-4 from Glycine max) or crude recombinant Escherichia coli cell lysates in 96-well microtiter plates. The suitability of the colorimetric assay for screening mutant GST variants derived from a directed evolution library was successfully evaluated. In addition, the assay was also used for screening GST synthetic inhibitors. It was concluded that the proposed colorimetric assay is selective and sensitive and allows the screening of large numbers of samples within a few minutes.
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PMID:A new colorimetric assay for glutathione transferase-catalyzed halogen ion release for high-throughput screening. 2052 59


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