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)

In an attempt to seek out new factors that are related to colorectal carcinogenesis at the molecular level, subtractive hybridization between cDNA of normal mucosal tissues and mRNA of colorectal carcinoma tissues was performed. Subsequent screenings of the cDNA libraries, constructed from normal mucosal tissues, using the "subtractive probes" generated a total of 46 clones that were expressed in normal mucosa but were either expressed at a significantly reduced level or not expressed at all in cancer tissues. Partial nucleotide sequences of all of these cDNA clones were determined, and sequence homology analyses were performed with the Genbank database. Of the 46 cDNA samples, 44 contained substantial sequence homologies with 32 immunoglobulin gene fragments, a helix-loop-helix basic phosphoprotein gene, an acidic ribosomal phosphoprotein P2 gene, a BLR1 gene for Burkitt's lymphoma receptor 1 gene, D5S419 DNA segment containing (C-A) repeats, a glucokinase (GCK) gene, a Na+, K+-ATPase alpha-subunit gene, a histocompatibility system HLA-DR heavy-chain gene, a dystrophic gene, a mucin (MUC2) gene, a mu-glutathione S-transferase gene, a Menkes disease protein gene, and a 40-kDa keratin intermediate filament precursor gene. The remaining two cDNA clones (now registered under GenBank accession numbers U17714 and U20428) showed few (less than 60%) sequence homologies with any known sequences in the GenBank database and, therefore, may represent novel genes whose expression was down-regulated in human colorectal carcinomas. The possible clinical significance of these findings and the involvement of these two genes in the carcinogenesis of colorectal as well as other cancers are being investigated.
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PMID:Characterization of colorectal-cancer-related cDNA clones obtained by subtractive hybridization screening. 929 8

The Drosophila nucleosome remodeling factor NURF utilizes the energy of ATP hydrolysis to perturb the structure of nucleosomes and facilitate binding of transcription factors. The ATPase activity of purified NURF is stimulated significantly more by nucleosomes than by naked DNA or histones alone, suggesting that NURF is able to recognize specific features of the nucleosome. Here, we show that the interaction between NURF and nucleosomes is impaired by proteolytic removal of the N-terminal histone tails and by chemical cross-linking of nucleosomal histones. The ATPase activity of NURF is also competitively inhibited by each of the four Drosophila histone tails expressed as GST fusion proteins. A similar inhibition is observed for a histone H4 tail substituted with glutamine at four conserved, acetylatable lysines. These findings indicate a novel role for the flexible histone tails in chromatin remodeling by NURF, and this role may, in part, be independent of histone acetylation.
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PMID:Role of histone tails in nucleosome remodeling by Drosophila NURF. 930 16

A 1.7-kilobase pair segment from the conjugative transfer region of plasmid R388 DNA was cloned and sequenced. It contained trwD, a gene essential for plasmid R388 conjugation, for expression of the conjugative W-pilus and for sensitivity to phage PRD1. The deduced amino acid sequence of TrwD showed homology to the PulE/VirB11 superfamily of potential ATPases involved in various types of transport processes. A fusion of trwD with the glutathione S-transferase (GST) was constructed, and the resulting fusion protein was purified from overproducing bacteria. Factor Xa hydrolysis of GST-TrwD and further purification rendered TrwD protein with more than 95% purity. Antibodies raised against TrwD localized it both in the soluble fraction and in the outer membrane of Escherichia coli. TrwD is probably a peripheral outer membrane protein because it could be solubilized by increasing salt concentration to 0.5 M NaCl in the lysis buffer. Both purified GST-TrwD and TrwD could hydrolize ATP. ATPase activity increased 2-fold in the presence of detergent-phospholipid mixed micelles. To study the importance of the nucleotide-binding site, Walker box A (GXXGXGK(T/S)), present in TrwD, the conserved lysine residue was replaced by glutamine. The mutant protein, expressed and purified under the same conditions as the wild type, did not exhibit ATPase activity. TrwD(K203Q) was not able to complement the mutation in trwD of the R388 mutant plasmid, suggesting the essentiality of the ATPase activity of the protein in the conjugative process. Furthermore, the dominant character of this mutation suggested that GST-TrwD(K432Q) was still able to interact either with itself or with other component(s) of the conjugative machinery.
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PMID:TrwD, a protein encoded by the IncW plasmid R388, displays an ATP hydrolase activity essential for bacterial conjugation. 932 77

Interactions of the F1F0-ATPase subunits between the cytoplasmic domain of the b subunit (residues 26-156, bcyt) and other membrane peripheral subunits including alpha, beta, gamma, delta, epsilon, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitro reconstitution of ATPase from the purified subunits as well. Only the combination of bcyt fused to the activation domain of the yeast GAL-4, and delta subunit fused to the DNA binding domain resulted in the strong expression of the beta-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of bcyt fused to glutathione S-transferase (GST) together with the delta subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-bcyt fusion alone had no such effect, indicating that GST-bcyt was protected by the co-expressed delta subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the delta subunit in the cell. The affinity purified GST-bcyt did not contain significant amounts of delta, suggesting that the interaction of these subunits was relatively weak. Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits. The ATPase activity was reconstituted from the purified bcyt together with alpha, beta, gamma, delta, and epsilon, or with the same combination except epsilon. Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex. The result indicated that interaction of b and delta was stabilized by F1 subunits other than epsilon and also suggested that b-delta interaction was important for F1-F0 interaction.
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PMID:Interaction of the delta and b subunits contributes to F1 and F0 interaction in the Escherichia coli F1F0-ATPase. 937 80

The putative copper binding domain from the copper-transporting ATPase implicated in Wilson disease (ATP7B) has been expressed and purified as a fusion to glutathione S-transferase. Immobilized metal ion affinity chromatography revealed that the fusion protein is able to bind to columns charged with different transition metals with varying affinities as follows: Cu(II)>>Zn(II)>Ni(II)>Co(II). The fusion protein did not bind to columns charged with Fe(II) or Fe(III). 65Zinc(II) blotting analysis showed that the domain is able to bind Zn(II) over a range of pH values from 6.5 to 9.0. Competition 65Zn(II) blotting showed that Cd(II), Hg(II), Au(III), and Fe(III) can successfully compete with Zn(II), at comparable concentrations, for binding to the domain. In contrast, the domain had little or no affinity for Ca(II), Mg(II), Mn(II), and Ni(II) relative to copper. Neutron activation analysis of the copper bound to the domain showed a copper:protein ratio of 6.5-7.3:1. Both Cu(II) and Cu(I) were found to have a higher affinity for the domain relative to Zn(II). In addition, a sharp, reproducible transition was only observed in competition experiments with copper, which may suggest that copper binding has some degree of cooperativity.
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PMID:Expression, purification, and metal binding properties of the N-terminal domain from the wilson disease putative copper-transporting ATPase (ATP7B). 940 18

Eukaryotic P-type ATPases use energy to drive the transport of cations across membranes. A complete P-ATPase gene (CpATPase1) has been isolated from Cryptosporidium parvum, one of the opportunistic pathogens in AIDS patients. The complete gene encodes 1528 amino acids, predicting a protein of 169 kDa. A hydropathy profile of the protein suggested there are eight transmembrane domains (TM). Expression of the gene was confirmed both by Northern blot analysis and RT-PCR. A fragment of the gene has been expressed as a 49 kDa GST-fusion protein. This protein was used to produce rabbit antiserum and fluorescent labeling has localized the protein to the sporozoite apical and perinuclear regions. SDS-PAGE and Western blot analysis show a 160 kDa major protein, close to the predicted size. The protein shares greatest overall identity and similarity to a putative organellar Ca2+ P-ATPase described for Plasmodium falciparum. Unlike P. falciparum, but consistent with all genes so far isolated from C. parvum, the gene contains no introns. The Ca2+ P-ATPases from these two Apicomplexa are large and do not have motifs predicting calmodulin-binding.
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PMID:Molecular analysis of a P-type ATPase from Cryptosporidium parvum. 949 52

A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H+-ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H+-ATPase. The binding was dependent on Mg2+ and was strongly increased by fusicoccin. Controlled trypsin digestion of H+-ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the C-terminal domain of H+-ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.
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PMID:Fusicoccin effect on the in vitro interaction between plant 14-3-3 proteins and plasma membrane H+-ATPase. 951 76

The mrsC gene of Escherichia coli is required for mRNA turnover and cell growth, and strains containing the temperature-sensitive mrsC505 allele have longer half-lives than wild-type controls for total pulse-labeled and individual mRNAs (L. L. Granger et al., J. Bacteriol. 180:1920-1928, 1998). The cloned mrsC gene contains a long open reading frame beginning at an initiator UUG codon, confirmed by N-terminal amino acid sequencing, encoding a 70,996-Da protein with a consensus ATP-binding domain. mrsC is identical to the independently identified ftsH gene except for three additional amino acids at the N terminus (T. Tomoyasu et al., J. Bacteriol. 175:1344-1351, 1993). The purified protein had a Km of 28 microM for ATP and a Vmax of 21.2 nmol/microg/min. An amino-terminal glutathione S-transferase-MrsC fusion protein retained ATPase activity but was not biologically active. A glutamic acid replacement of the highly conserved lysine within the ATP-binding motif (mrsC201) abolished the complementation of the mrsC505 mutation, confirming that the ATPase activity is required for MrsC function in vivo. In addition, the mrsC505 allele conferred a temperature-sensitive HflB phenotype, while the hflB29 mutation promoted mRNA stability at both 30 and 44 degrees C, suggesting that the inviability associated with the mrsC505 allele is not related to the defect in mRNA decay. The data presented provide the first direct evidence for the involvement of a membrane-bound protein in mRNA decay in E. coli.
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PMID:Escherichia coli mrsC is an allele of hflB, encoding a membrane-associated ATPase and protease that is required for mRNA decay. 953 94

Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 microM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis-Menten kinetics (Km 46 microM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis-Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 microM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 microM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 microM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen.
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PMID:ATP-dependent transport of glutathione conjugate of 7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles. 962 Aug 85

Deng and Shuman (J. Biol Chem. 271, 29386 (1996)) reported that an ATPase different from the known viral termination factor, VTF, is required for vaccinia virus early gene transcription termination. Properties of this ATPase were similar to those of a known vaccinia virus enzyme, nucleoside triphosphate phosphohydrolase I (NPH I) the product of gene D11L. Transcription-competent cell-free extracts were prepared from A549 cells infected with wild-type or mutant vaccinia virus harboring ts mutations in gene D11L. These extracts were employed to investigate the role of NPH I in early gene transcription termination. Extracts prepared under nonpermissive conditions from both wild-type virus and ts mutant virus-infected cells exhibited high levels of early and intermediate gene transcription activity but were incapable of supporting late gene transcription. ts mutant extract lacked signal-dependent early gene transcription termination activity, which was restored by the addition of either free NPH I or a GST-NPH I fusion protein. A comparison of the NPH I amino acid sequence to the protein databases revealed the presence of a set of sequences characteristic of nucleic acid helicase superfamily II members. A series of site-specific mutations in the helicase motifs and N-terminal and C-terminal deletion mutations were expressed as GST fusion proteins and their activities assessed. Of the mutations in helicase motifs I to VI, alteration of all but motif III reduced the ATPase activity. Removal of as few as 24 amino acids from the N-terminal end eliminated ATPase activity, while deletion of 68 C-terminal amino acids exhibited only a modest decrease in ATP hydrolysis. Larger C-terminal deletions eliminated ATPase activity. Each deletion mutation, and site-specific mutations other than the motif III mutation, failed to support transcription termination in vitro. Mutations in motifs I, II, V, and VI inhibit wild-type NPH I transcription termination activity. However, deletion of up to 68 amino acids from the C-terminal end eliminates this inhibitory property. This observation is particularly interesting since these C-terminal deletions retain both ATPase activity and single-stranded DNA binding activity. Their failure to inhibit transcription termination suggests that these C-terminal deletion mutations eliminate a site required for a function other than from DNA binding or ATP hydrolysis.
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PMID:Vaccinia virus nucleoside triphosphate phosphohydrolase I is an essential viral early gene transcription termination factor. 963 76


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