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)

This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced.
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PMID:Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems. 760 44

We previously demonstrated that Treponema pallidum cells incubated in vitro in the presence of heat-inactivated normal rabbit serum (HINRS) synthesize, in very small quantities, several pathogen-specific, low-molecular-mass proteins that appear to be localized extracellularly. In this study, we have taken advantage of our ability to metabolically radiolabel T. pallidum cells to high specific activity to further characterize these antigens. We found that the low-molecular-mass proteins are not related to the 15- and 17-kDa detergent-phase proteins (J. D. Radolf, N. R. Chamberlain, A. Clausell, and M. V. Norgard, Infect. Immun. 56:490-498, 1988). The low-molecular-mass proteins did not incorporate 3H-labeled fatty acids and were not precipitated by rabbit immunoglobulin G (IgG) antibodies directed against glutathione S-transferase fusions to the nonlipidated 15- and 17-kDa proteins. We prepared polyclonal antisera to the low-molecular-mass proteins by immunizing two rabbits with the concentrated supernatant of T. pallidum cells. IgG antibodies present in the sera of both rabbits precipitated a 21.5-kDa protein from solubilized extracts of T. pallidum supernatant and cells. IgG antibodies in the serum of the second rabbit precipitated an additional 15.5-kDa low-molecular-mass protein only from solubilized extracts of supernatant. While investigating the effect of eliminating HINRS from the extraction medium, we observed that the low-molecular-mass proteins remained associated with treponemal cells that were incubated in the absence of HINRS. These proteins could be eluted from the cells by the addition of HINRS or rabbit serum albumin, suggesting that they are located on or near the treponemal cell surface. The 15.5- and 21.5-kDa low-molecular-mass proteins were not washed off treponemal cells with buffer containing 1 M KCl. Experiments employing selective solubilization of the T. pallidum outer membrane with 0.1% Triton X-114 and proteinase K accessibility indicated that the 15.5-kDa protein, but not the 21.5-kDa protein, is cell surface exposed.
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PMID:Characterization of the low-molecular-mass proteins of virulent Treponema pallidum. 826 39

PrP27-30 represents the protease-resistant core of the prion protein and was found to be the main component in Scrapie prion preparations. Recombinant (r) PrP27-30 corresponding to aa 90-231 from the Syrian golden hamster prion protein was expressed as a fusion with GST in E. coli and secreted from insect cells infected with recombinant baculoviruses, GST::rPrP27-30 isolated from either system was purified to homogenity by glutathione-Sepharose chromatography. rPrP27-30 from both systems was generated by direct cleavage of GST::rPrP27-30 in the presence of thrombin revealing a molecular weight of 17 kDa. GST::rPrP27-30 as well as the authentic protein rPrP27-30 were identified by immunoblotting employing a polyclonal antibody directed against a peptide corresponding to aa 95-110 of the Syrian golden hamster prion protein. In contrast to scrapie prior PrP27-30, the recombinant proteins GST::rPrP27-30 and rPrP27-30 were both sensitive towards proteinase K, suggesting that the molecules lack infectivity.
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PMID:Recombinant prion protein rPrP27-30 from Syrian golden hamster reveals proteinase K sensitivity. 861 3

Until quite recently, high-level expression of full-length cellular prion protein (Prp(c)) in bacterial cells was not possible. We describe here the effective purification of mature Syrian golden hamster PrPc (residues 23-231) as a C-terminal fusion to glutathione S-transferase (GST) from inclusion bodies expressed in Escherichia coli. Purification of the denatured fusion protein was simplified greatly by the introduction of a C-terminal histidine anchor, leading to 255 mg pure GST-PrPc-His6/l bacterial broth, which could be refolded easily by dilution in 20 mM Tris, 5 mM dithiothreitol, 1 mM EDTA, pH 9.0. Refolding was monitored by following GST activity. Mature Syrian hamster PrPc (residues 23-231) was released from the refolded fusion protein by thrombin digestion, yielding 73 mg homogeneous protein/l bacterial culture after purification. The recombinant protein was identified by monoclonal antibodies, Edman sequencing and matrix-assisted laser-desorption/ionization MS. Correct folding was confirmed by near-ultraviolet circular dichroism spectroscopy. Samples resulting from different purification steps were sensitive to proteinase K digestion and showed no signs of infectivity in animal experiments, demonstrating that the PrPc produced is identical with the cellular isoform. The presented purification procedure should prove useful for the production of other GST-fusion proteins.
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PMID:Large-scale production, purification and refolding of the full-length cellular prion protein from Syrian golden hamster in Escherichia coli using the glutathione S-transferase-fusion system. 949 19

A 1116 bp open reading frame (ORF), designated jlpA, encoding a novel species-specific lipoprotein of Campylobacter jejuni TGH9011, was identified from recombinant plasmid pHIP-O. The jlpA gene encodes a polypeptide (JlpA) of 372 amino acid residues with a molecular mass of 42.3 kDa. JlpA contains a typical signal peptide and lipoprotein processing site at the N-terminus. The presence of a lipid moiety on the JlpA molecule was confirmed by the incorporation of [3H]-palmitic acid. Immunoblotting analysis of cell surface extracts prepared using glycine-acid buffer (pH 2.2) and proteinase K digestion of whole cells indicated that JlpA is a surface-exposed lipoprotein in C. jejuni. JlpA is loosely associated with the cell surface, as it is easily extracted from the C. jejuni outer membrane by detergents, such as sarcosyl and Triton X-100. JlpA is released to the culture medium, and its concentration increases in a time-dependent fashion. The adherence of both insertion and deletion mutants of jlpA to HEp-2 epithelial cells was reduced compared with that of parental C. jejuni TGH9011. Adherence of C. jejuni to HEp-2 cells was inhibited in a dose-dependent manner when the bacterium was preincubated with anti-GST-JlpA antibodies or when HEp-2 cells were preincubated with JlpA protein. A ligand-binding immunoblotting assay showed that JlpA binds to HEp-2 cells, which suggests that JlpA is C. jejuni adhesin.
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PMID:JlpA, a novel surface-exposed lipoprotein specific to Campylobacter jejuni, mediates adherence to host epithelial cells. 1125 39

The transcription factor NF-kappaB regulates a wide set of genes involved in the establishment of many cellular processes that control cell activation, proliferation, and apoptosis. IkappaB inhibitory subunits integrate NF-kappaB activation signals through phosphorylation and ubiquitination of its N-terminal domain. Using the two-hybrid system in yeast, we searched for IkappaB-alpha N-terminal domain interactors and therefore potential NF-kappaB regulators. An interaction of IkappaB-alpha with the mitochondrial ATP/ADP translocator ANT was detected in yeast and confirmed in glutathione S-transferase pull-down assays and co-precipitation experiments in transfected cells. Subcellular cell fractionation, resistance to proteinase K treatment, and electron microscopy experiments demonstrated the presence of IkappaB-alpha and associated p65 NF-kappaB in the mitochondrial intermembrane space. IkappaB-alpha.NF-kappaB appeared to be released from mitochondria upon the induction of apoptosis by engagement of the Fas receptor. These data suggest that the mitochondrial IkappaB-alpha.NF-kappaB pool participates in the regulation of apoptosis.
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PMID:Ikappa b-alpha, the NF-kappa B inhibitory subunit, interacts with ANT, the mitochondrial ATP/ADP translocator. 1128 11

This study evaluates the influence of genetic polymorphism at GSTM1, GSTM3 and GSTT1 gene loci on oral cancer risk among Indians habituated to the use of, smokeless tobacco, bidi or cigarette. DNA extracted from white blood cells of 297 cancer patients and 450 healthy controls by the proteinase K phenol-chloroform extraction procedure were analyzed by the polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) analyses. Lifetime tobacco exposure was evaluated as a risk factor in relation to the polymorphism at the GST gene loci using logistic regression analysis. There was no significant difference in the distribution of the GSTM3 and GSTT1 genotypes between oral cancer patients and controls. In contrast, a significant 3-fold increase in risk was seen for patients with the GSTM1 null genotype (age adjusted OR = 3.2, 95% CI 2.4-4.3). The impact of the GSTM1 null genotype on oral cancer risk was also analyzed in separate groups of individuals with different tobacco habits. The odds ratio associated with the GSTM1 null genotype was 3.7 (95% CI 2.0-7.1) in tobacco chewers, 3.7 (5% CI 1.3-7.9) in bidi smokers and 5.7 (95% CI 2.0-16.3) in cigarette smokers. Furthermore, increased lifetime exposure to chewing tobacco appeared to be associated with a 2-fold increase in oral cancer risk in GSTM1 null individuals. The results suggest that the GSTM1 null genotype is a risk factor for development of oral cancer among Indian tobacco habitues.
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PMID:Polymorphism at GSTM1, GSTM3 and GSTT1 gene loci and susceptibility to oral cancer in an Indian population. 1201 53

The glutathione S-transferase (GST) family of enzymes has a vital role in phase II of biotransformation of environmental carcinogens, pollutants, drugs and other xenobiotics. GSTs are polymorphic, with the type and frequency of polymorphism being ethnic dependent. Polymorphisms in GST genes have been shown to be associated with susceptibility to disease and disease outcome. We determined the frequencies of GSTM1, GSTT1 and GSTP1 polymorphisms in 591 volunteers who had been residents of Rio de Janeiro for at least six months. Blood was collected and DNA extracted by proteinase K/SDS digestion. Information about social habits and health problems was also recorded. GSTM1 and GSTT1 polymorphisms were analyzed by a PCR-Multiplex procedure, whereas GSTP1 polymorphism was analyzed by PCR-RFLP. We found that 42.1% (48.9% of whites and 34.2% of non-whites) of the individuals had the GSTM1 null genotype, whereas 25.4% (25.1% of whites and 25.7% of non-whites) had the GSTT1 null genotype. The genotypic distribution of GSTP1 was 49.7% I/I, 38.1% I/V, and 12.2% V/V, whereas the allelic frequencies were 0.69 for the Ile allele, and 0.31 for the Val allele. The frequencies of GST polymorphisms in this Brazilian population were found to be different from those observed in other populations, particularly of other South American countries.
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PMID:Frequencies of GSTM1, GSTT1, and GSTP1 polymorphisms in a Brazilian population. 1496 30

The trace mineral vanadium inhibits cancer development in a variety of experimental animal models. The present study was to gain insight into a putative anticancer effect of vanadium in a rat model of colon carcinogenesis. The in vivo study was intended to clarify the effect of vanadium on DNA-protein cross-links (DPC), surface level changes of aberrant crypt foci (ACF) and biotransformation status during 1,2-dimethylhydrazine (1,2-DMH) induced preneoplastic rat colon carcinogenesis. The comet assay showed statistically higher mean base values of DNA-protein mass (p<0.01) and mean frequencies of tailed cells (p<0.001) in the carcinogen-induced group after treatment with proteinase K. Treatment with vanadium in the form of ammonium monovanadate supplemented ad libitum in drinking water for the entire experimental period caused a significant (p<0.02) reduction (40%) in DNA-protein cross-links in colon cells. Further, the biotransformation status of vanadium was ascertained measuring the drug metabolising enzymes, glutathione S-transferase (GST) and cytochrome P-450 (Cyt P-450). Significantly, there was an increase in glutathione S-transferase and cytochrome P-450 levels (p<0.01 and p<0.02, respectively) in rats supplemented with vanadium as compared to their carcinogen controls. As an endpoint marker, we also evaluated the effect of vanadium on surface level changes of aberrant crypt foci induced by 1,2-DMH by scanning electron microscopy. Animals induced with 1,2-DMH and supplemented with vanadium showed a marked improvement in colonic architecture with less number of aberrant crypt foci in contrast to the animals induced with 1,2-DMH alone, thereby exhibiting its anti-carcinogenicity by modulating the markers studied herein.
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PMID:Vanadium inhibits DNA-protein cross-links and ameliorates surface level changes of aberrant crypt foci during 1,2-dimethylhydrazine induced rat colon carcinogenesis. 1586 87

Bacterial recognition of host sialic acid-containing receptors plays an important role in microbial colonization of the human oral cavity. The aggregation of human platelets by Streptococcus gordonii DL1 is implicated in the pathogenesis of infective endocarditis. In addition, we consider that hemagglutination of this organism may act as an additive factor to increase the severity of this disease. We previously reported that this interaction requires the bacterial expression of a 203-kDa protein (Hsa), which has sialic acid-binding activity. In the present study, we confirmed that erythrocyte surface sialoglycoproteins are the receptors for Hsa. We examined the effects of proteinase K, chymotrypsin, phospholipase C, and alpha(2-3) or alpha(2-3, 6, 8) neuraminidase on hemagglutination activity and found that the interaction occurs between Hsa and alpha2-3-linked sialic acid-containing proteins of erythrocytes. We expressed recombinant NR2, which is the putative binding domain of Hsa, fused with GST in Escherichia coli BL21. Dot-blot analysis demonstrated that GST-HsaNR2 binds both glycophorin A (GPA) and band 3. Moreover, GPA and a small amount of band 3 were detected by GST pull-down assays. These findings indicate that S. gordonii Hsa specifically binds to GPA and band 3, alpha2-3-linked sialic acid membrane glycoproteins.
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PMID:Hsa, an adhesin of Streptococcus gordonii DL1, binds to alpha2-3-linked sialic acid on glycophorin A of the erythrocyte membrane. 1838 Aug 4


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