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:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.
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PMID:Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase. 879 93

We cloned and sequenced the second open reading frame of the RNA polymerase gene, ORF1b, of bovine coronavirus. In the region representing nucleotide positions 4919-5677 upstream from the initiation codon of the 32K non-structural protein gene, we identified two putative functional domains. One of these domains contained four leucine residues repeated exactly in every seventh position, and the other domain represented a cluster of cysteine and histidine residues. The DNA sequence representing these domains was cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. A high level expression of the cysteine-rich domain was achieved as a fusion protein when the bacterial culture was induced with IPTG. In a solid phase zinc binding assay using the recombinant fusion protein, we found that the protein containing the cysteine-rich domain was able to bind to radioactive zinc in vitro, demonstrating that the polypeptide encoded by the ORF1b of coronavirus is a zinc-binding protein.
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PMID:Zinc-binding of the cysteine-rich domain encoded in the open reading frame of 1B of the RNA polymerase gene of coronavirus. 883 May 21

The cuticle of parasitic nematodes is composed of extracellular structural proteins. Over 90% of these proteins are collagenous molecules in the basal and median layers of the cuticle. The outermost layers of the cuticle, the epicuticle, is composed of non-collagenous proteins, that represent the structural surface of nematodes. In Ascaris these proteins have been termed 'cuticlins'. While cuticular collagens have been well studied by both biochemical and genetic means, knowledge of the molecular structure of cuticlin components of parasitic nematodes is scarce. In the present paper we report on the production of monoclonal antibody 8.1, which is specific for cuticlin, but does not recognize collagen epitopes. We have screened a cDNA library derived from adult Ascaris suum mRNA of the hypodermal tissue underlying and synthesizing the cuticle. One positive cDNA clone encodes alanine-rich repetitive motifs, which are part of the insoluble cuticlin of the outermost layers of the epicuticle of Ascaris suum. This was shown in immunocytochemical experiments using specific polyclonal antisera raised against these motifs, expressed as fusion protein with glutathione S-transferase of the helminth Schistosoma japonicum. Comparison of the repetitive amino acid sequence with structural proteins of the nematode Caenorhabditis elegans and the insects Locusta migratoria and Ceratitis capitata revealed a minimal consensus motif.
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PMID:Repetitive peptide motifs in the cuticlin of Ascaris suum. 888 22

To determine the location of the non-substrate-ligand-binding region in mammalian glutathione S-transferases, fluorescence-resonance energy transfer was used to calculate distances between tryptophan residues and protein-bound 8-anilinonaphthalene 1-sulphonate (an anionic ligand) in the human class-alpha glutathione S-transferase, and in a human Trp28-->Phe mutant class-pi glutathione S-transferase. Distance values of 2.21 nm and 1.82 nm were calculated for the class-alpha and class-pi enzymes, respectively. Since glutathione S-transferases bind one non-substrate ligand/protein dimer, the ligand-binding region, according to the calculated distances, is found to be located in the dimer interface near the twofold axis. This region is the same as that in which the parasitic helminth Schistosoma japonicum glutathione S-transferase binds praziquantel, a non-substrate drug used to treat schistosomiasis [McTigue, M. A., Williams, D. R. & Tainer, J. A. (1995) J. Mol. Biol. 246, 21-27]. Since the overall folding topology is conserved and certain features at the dimer interface are similar throughout the superfamily, it is reasonable to expect that all cytosolic glutathione S-transferases bind non-substrate ligands in the amphipathic groove at the dimer interface.
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PMID:Determination of a binding site for a non-substrate ligand in mammalian cytosolic glutathione S-transferases by means of fluorescence-resonance energy transfer. 891 46

Six monoclonal antibodies (MAbs) were raised in mice against the 26-kDa glutathione S-transferase (GST) of the parasite Schistosoma japonicum. These MAbs were originally selected for their specific binding to the recombinant GST (r-GST) generated in E. coli by an enzyme-linked immunosorbent assay. A further study demonstrated that all these MAbs bound to plate-coated GST affinity-purified from the parasite Schistosoma japonicum. However, in Western blotting analysis only a single monoclonal antibody (MAb Y3D7) yielded positive binding. The binding of MAb Y3D7 on Western blotting was further characterized; specific binding was found on other GST fusion proteins and on the authentic 26-kDa GST but not the 28-kDa GST in the total soluble worm proteins from Schistosoma japonicum. Using protein-A-mediated immunoprecipitation, MAbs Y3D7 and Y5D5 precipitated r-GST while in parallel experiments the remaining MAbs did not generate r-GST precipitation. In an alternative co-precipitation experiment, r-GST was first bound to glutathione (GSH) Sepharose beads and subsequently tested for interaction with the MAbs. In this manner, all MAbs except MAb Y5D5 were co-precipitated with the complexes. Thus, these select MAbs readily reacted with GST although their binding characteristics were different. Because GST has been widely used in the generation of fusion proteins for various purposes and is a potential vaccine candidate in controlling schistosomiasis, these MAbs should prove valuable for their application to molecular biology and parasitology.
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PMID:Characterization of monoclonal antibodies to the 26-kDa glutathione S-transferase of Schistosoma japonicum. 898 54

Human c-Jun and c-Fos leucine zipper domains were examined for their ability to serve as autonomous dimerization domains as part of a heterologous protein construct. Schistosoma japonicum glutathione S-transferase (GST) was fused to recombinant Jun leucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains. SDS-PAGE 'snapshot' analyses based on disulphide linkage of monomers demonstrated the ability of rJunLZ to function as a dimerization motif in a foreign protein environment. Steric hindrance prevented formation of rJunLZ-GST::rFosLZ-GST heterodimers whereas rJunLZ-GST::rFosLZ and rJunLZ:: rFosLZGST formed readily. Furthermore, rJunLZGST generated homodimers suggesting fusion protein heterodimers interact differently to homodimers. Gel filtration chromatography confirmed that GST is a dimer in solution and that attachment of a leucine zipper domain allows further interactions to take place. Sedimentation equilibrium analyses showed that GST is a stable dimer (K(a) > 10(6) M(-1)) with no higher multimeric forms. rFosLZ-GST weakly associates beyond a dimer (K(a) approximately 4 x 10(4) M(-1)) and rJunLZ-GST associates indefinitely (K(a) approximately 4 x 10(5) M(-1)) [corrected], consistent with an isodesmic model of association. The interaction of these leucine zippers independently of GST association demonstrates their utility in the modification of proteins when multimer formation is desired.
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PMID:Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module. 900 44

The Sulfolobus solfataricus, strain MT4, beta-glycosidase (Ss beta-gly) is a thermophilic member of glycohydrolase family 1. To identify active-site residues, glutamic acids 206 and 387 have been changed to isosteric glutamine by site-directed mutagenesis. Mutant proteins have been purified to homogeneity using the Schistosoma japonicum glutathione S-transferase (GST) fusion system. The proteolytic cleavage of the chimeric protein with thrombin was only obtainable after the introduction of a molecular spacer between the GST and the Ss beta-gly domains. The Glu387-->Gln mutant showed no detectable activity, as expected for the residue acting as the nucleophile of the reaction. The Glu206-->Gln mutant showed 10- and 60-fold reduced activities on aryl-galacto and aryl-glucosides, respectively, when compared with the wild type. Moreover, a significant Km decrease with p/o-nitrophenyl-beta-D-glucoside was observed. The residual activity of the Glu206-->Gln mutant lost the typical pH dependence shown by the wild type. These data suggest that Glu206 acts as the general acid/base catalyst in the hydrolysis reaction.
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PMID:Identification of two glutamic acid residues essential for catalysis in the beta-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus. 901 Sep 32

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol.
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PMID:Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag. 904 42

The gene encoding Mhp1, a 124 kDa protein from Mycoplasma hyopneumoniae, has been cloned, sequenced, and its product characterized. No significant homology to the gene or encoded polypeptide was found in the Genbank, NBRF, or PIR databases, though this protein appears similar to p97, a putative adhesin of M. hyopneumoniae described by Zhang et al. (Infect. Immun. 63, 1013-1019, 1995). Two repeated motifs were identified within the 3' end of the gene and encoded polypeptide. The mhp1 gene was fused to the glutathione S-transferase (GST) gene from Schistosoma japonicum, enabling high-level expression and purification of the protein. Both the authentic and recombinant proteins were recognized by sera from pigs infected with M. hyopneumoniae. In an induced-disease model in pigs, coughing was reduced in animals vaccinated with recombinant GST-Mhp1, although differences were not significant. Only minimal protection against lung lesion formation was provided, and again differences between the Mhpl-vaccinated and nonvaccinated groups were not significant.
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PMID:Characterization of the gene encoding Mhp1 from Mycoplasma hyopneumoniae and examination of Mhp1's vaccine potential. 904 63

We have shown previously that immunisation of mice and pigs with recombinant 26 kDa GST (reSjc26GST) induces a pronounced anti-fecundity effect after experimental infection with Chinese Schistosoma japonicum. We report here that anti-fecundity immunity can also be induced against reSjc26GST in Chinese water buffaloes (Bos buffelus), important reservoir hosts for S. japonicum in China. Anti-Sjc26GST antibodies were produced in immunised buffaloes and, following challenge with S. japonicum cercariae, a 22.3% reduction in worm numbers was evident in vaccinated when compared with control animals. The anti-fecundity effect was characterised by a significant decrease in faecal egg output and eggs deposited in host tissues with those in the liver and intestine being reduced by about 50%. In addition to the anti-fecundity effect, reSjc26GST reduced by nearly 40% the egg-hatching capacity of S. japonicum eggs into viable miracidia. In terms of vaccination strategy, these effects would combine to diminish pathology in animals immunised with reSjc26GST and reduce transmission of schistosomiasis japonica.
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PMID:Anti-fecundity immunity to Schistosoma japonicum induced in Chinese water buffaloes (Bos buffelus) after vaccination with recombinant 26 kDa glutathione-S-transferase (reSjc26GST). 918 28


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