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)

The temporal effects of vitamin A deficiency on hepatic cytochrome P-450-dependent and conjugation reactions were studied in the rat. Cytochrome P-450 levels and N-methyl-p-chloroaniline N-demethylase activity were significantly reduced in the deficient animals. No other changes in parameters dependent on cytochrome P-450 were observed in vitro. Decreases in hepatic cytochrome P-450 were accompanied by a prolongation in hexobarbital sleeping times in deficient animals. The p-aminobenzoic acid N-acetyltransferase activity was higher in the deficient animals at 8 weeks, but by 10 weeks the activity in fact was significantly lower as compared to controls. Activities of 'native' and UDP N-acetylglucosamine 'activated' UDP-glucuronyltransferase were reduced in vitamin A deficiency. In contrast to this general pattern of impaired drug metabolism in vitamin A deficiency, glutathione S-aryltransferase activity was markedly enhanced at all time points from 4 to 10 weeks. Activities of this enzyme were twice controls at 6 weeks, a time at which no other enzyme changes were observed.
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PMID:Hepatic cytochrome P-450-dependent metabolism and enzymatic conjugation of foreign compounds in vitamin A-deficient rats. 722 May 90

The recognition of lysosomal enzymes by UDP-GlcNAc: lysosomal-enzyme GlcNAc-1-phosphotransferase (phosphotransferase) is mediated by a protein structure on lysosomal enzymes. It has been previously demonstrated that lysine residues are required for phosphorylation of procathepsin L and are a common feature of the site on many lysosomal proteins. In this work, the procathepsin L recognition structure was further defined by identification of the region of the protein containing the structure and the critical lysine residues involved. Removal of the cathepsin L propeptide by low pH-induced autocatalytic processing abolished phosphorylation. The addition of either the purified propeptide or a glutathione S-transferase-propeptide fusion protein to the processed protein restored phosphorylation. Mutagenesis of individual lysine residues demonstrated that two propeptide lysine residues (Lys-54 and Lys-99) were required for efficient phosphorylation of procathepsin L. By comparison of the phosphorylation rates of procathepsin L, lysine-modified procathepsin L, and the procathepsin L oligosaccharide, lysine residues were shown to account for most, if not all, of the protein-dependent interaction. On this basis, it is concluded that the proregion lysine residues are the major elements of the procathepsin L recognition site. In addition, lysine residues in cathepsin D were shown to be as important for phosphorylation as those in procathepsin L, supporting a general model of the recognition site as a specific three-dimensional arrangement of lysine residues exposed on the surface of lysosomal proteins.
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PMID:Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation. 779 59

A photoreactive N-acetylglucosamine derivative, N-[2-[2-[2-(2-biotinylaminoethoxy)-ethoxy]ethoxy]-4-[3-(trifluo rom ethyl)-3-H-diazirin-3-yl]benzoyl]-N4-[2-(acetylamino)-deoxy-beta-D -glucopyranosyl]-L-aspartamide (BDGA), was synthesized as a carbene-generating biotinylated probe for UDP-galactose:N-acetylglucosamine beta-(1-->4)-galactosyltransferase (GalT). The photoaffinity labeling experiments of bovine GalT with BDGA under various condition were examined based on the quantitative chemiluminescent detection of the biotinyl residue which was photochemically introduced into in GalT protein. A progressive decrease in the yield of specific photolabeling was observed upon lowering the incubation temperature from 37 degrees C to 20 degrees C or 4 degree C. The amount of photoincorporation was also decreased when UMP was not included in the incubation mixture. Using a crude protein mixture of recombinant human GalT, a band corresponding to the glutathione S-transferase fusion GalT protein was also specifically visualized. Furthermore, combine use of BDGA photolabeling with an immobilized avidin was found to be effective for the selective retrieval of photolabeled GalT from a reaction mixture containing a large amount of unlabeled GalT protein. The results obtained clearly demonstrate that the covalent biotinylation using the carbene-generating photoaffinity reagent BDGA would be useful for the analysis of acceptor substrate binding sites within the GalT protein.
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PMID:Synthesis and characterization of a carbene-generating biotinylated N-acetylglucosamine for photoaffinity labeling of beta-(1-->4)-galactosyltransferase. 901 80

To identify and analyze acceptor sequences for O-glycosylation, we have developed an in vivo system expressing short peptides as glutathione S-transferase fusion proteins in the eukaryotic host Dictyostelium discoideum. Using this approach, we show that a short peptide motif (PTVTPT), present in the D. discoideum cell-surface glycoprotein PsA, is sufficient as a signal for O-glycosylation, even when fused to a heterologous protein. Monosaccharide analysis and solid-phase protein sequencing showed that the modification is a single N-acetylglucosamine attached to threonine residues. This was further confirmed by electrospray-mass spectrometry. The O-linked glycosylation of both this peptide and authentic PsA presents the modB-dependent carbohydrate-specific epitope identified by the monoclonal antibody MUD50. Substitution of threonine by serine residues in this peptide also yields a glycosylated fusion protein which is modified with single N-acetylglucosamine residues, but not all of the serines are glycosylated.
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PMID:An in vivo approach for the identification of acceptor sites for O-glycosyltransferases: motifs for the addition of O-GlcNAc in Dictyostelium discoideum. 909 34

The transcription factor Sp1 plays an important role in the expression of many cellular genes. In studies of proteins that associate with Sp1, a 62-kDa glycoprotein was found in immunoprecipitates of Sp1. This protein was detected in these immunoprecipitates by the monoclonal antibody, RL2, which was originally raised against nuclear pore proteins but was subsequently found to recognize an epitope that contains O-linked N-acetylglucosamine (O-GlcNAc). The association of this protein with Sp1 could be blocked by SDS denaturation of the protein complex. Western blot analysis of the Sp1 immunoprecipitate using antibodies to p62 nucleoporin indicated that this nuclear pore protein associates with Sp1. Furthermore, immunoprecipitation of p62 nucleoporin resulted in the coprecipitation of Sp1. Recombinant p62, expressed as a GST-fusion protein using a vaccinia virus system, also interacted with both recombinant and native Sp1. This interaction between p62 and Sp1 required the C-terminus of p62 and the C-terminus was able to bind Sp1, albeit less efficiently than native p62. A mammalian two-hybrid interaction assay was devised in which p62 was fused to the Gal4 DNA-binding domain. This system also indicated that p62, through its C-terminus, interacts with Sp1 in the living cell. We propose that this interaction of a nuclear pore protein with Sp1 may reflect the nuclear organization required to bring transcribable DNA in contact with the transcription factors.
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PMID:Interaction of the transcription factor Sp1 with the nuclear pore protein p62 requires the C-terminal domain of p62. 940 13

In the present experiments the cDNA coding for a truncated form of the beta1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67-74-kDa fusion protein. Typically a yield of 750 microg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc, converting the acceptor to the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Accordingly, we call the recombinant beta1,6-GlcNAc transferase cIGnT6 to emphasize its action at central rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, beta1,4galactosyltransferase and beta1, 3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.
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PMID:The centrally acting beta1,6N-acetylglucosaminyltransferase (GlcNAc to gal). Functional expression, purification, and acceptor specificity of a human enzyme involved in midchain branching of linear poly-N-acetyllactosamines. 976 98

A procedure for the enzymatic synthesis of neoglycoenzymes is described. The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was overexpressed in Escherichia coli as a fusion protein linked to glutathione S-transferase (GST). GST-Endo-A fusion was extracted as a soluble protein. The fusion protein was purified to homogeneity with glutathione-Sepharose 4B and showed transglycosylation activity toward high-mannose-type glycopeptides without removing the GST moiety. The GST-Endo-A immobilized on glutathione-Sepharose 4B retained its transglycosylation activity. The immobilized enzyme could transfer (Man)(6)GlcNAc en bloc to partially deglycosylated ribonuclease B without damaging its enzyme activity. The immobilized GST-Endo-A should be very useful for synthesizing active neoglycoenzymes attached with homogeneous N-linked oligosaccharides.
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PMID:Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-beta-N-acetylglucosaminidase A. 1062 87

Protein glycosylation pathways are relatively poorly characterized in insect cells. As part of an overall effort to address this problem, we previously isolated a cDNA from Sf9 cells that encodes an insect alpha1,2-mannosidase (SfManI) which requires calcium and is inhibited by 1-deoxymannojirimycin. In the present study, we have characterized the substrate specificity of SfManI. A recombinant baculovirus was used to express a GST-tagged secreted form of SfManI which was purified from the medium using an immobilized glutathione column. The purified SfManI was then incubated with oligosaccharide substrates and the resulting products were analyzed by HPLC. These analyses showed that SfManI rapidly converts Man(9)GlcNAc(2)to Man(6)Glc-NAc(2)isomer C, then more slowly converts Man(6)GlcNAc(2)isomer C to Man(5)GlcNAc(2). The slow step in the processing of Man(9)GlcNAc(2)to Man(5)GlcNAc(2)by SfManI is removal of the alpha1,2-linked mannose on the middle arm of Man(9)GlcNAc(2). In this respect, SfManI is similar to mammalian alpha1,2-mannosidases IA and IB. However, additional HPLC and(1)H-NMR analyses demonstrated that SfManI converts Man(9)GlcNAc(2)to Man(5)GlcNAc(2)primarily through Man(7)GlcNAc(2)isomer C, the archetypal Man(9)GlcNAc(2)missing the lower arm alpha1,2-linked mannose residues. In this respect, SfManI differs from mammalian alpha1,2-mannosidases IA and IB, and is the first alpha1,2-mannosidase directly shown to produce Man(7)GlcNAc(2)isomer C as a major processing intermediate.
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PMID:N-Glycan processing by a lepidopteran insect alpha1,2-mannosidase. 1076 22

In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), Asp(278), and Arg(281) in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Delta null mutant. Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) did not. These results indicate that Ser(64), Asp(278), and Arg(281) of HsAgm1p are residues essential for the catalysis. Because Ser(64) corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser(64). Furthermore, alanine substitution for Arg(496) in region III significantly increased the K(m) value for N-acetylglucosamine-6-phosphate, demonstrating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phosphate.
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PMID:Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis. 1100 9

Like bacteria and many fungi, the pathogenic fungus Candida albicans can utilize GlcNAc as a carbon source for growth. A cluster of six genes was identified in the C. albicans genome. One of the genes in the cluster was CaNAG1, which is responsible for GlcN6P deaminase and is therefore essential for GlcNAc-dependent growth. The other five genes were designated CaNAG2, CaNAG3, CaNAG4, CaNAG5 and CaNAG6. The mRNA levels of CaNAG1, CaNAG2 and CaNAG5 were significantly induced by GlcNAc, whereas those of CaNAG3, CaNAG4 and CaNAG6 were not. Neither CaNAG2 nor CaNAG5 was essential for growth, but disruption of CaNAG2 or CaNAG5 greatly retarded the growth of cells using GlcNAc as the sole carbon source. Although no homolog of CaNAG2 or CaNAG5 was found in the Saccharomyces cerevisiae genome, CaNag2p displayed sequence similarities to Escherichia coli nagA, and CaNag5p is homologous to a wide variety of hexose kinases. When expressed as a fusion protein with glutathione S-transferase (GST), CaNag5p produced GlcNAc-P from GlcNAc in the presence of ATP, whereas GST alone did not. Furthermore, the recombinant GST-CaNag2p fusion protein converted GlcNAcP, which was produced by CaNag5p, into GlcNP. These results clearly demonstrate that CaNAG2 and CaNAG5 encode GlcNAcP deacetylase and GlcNAc kinase, respectively. CaNag5p recognized glucose and mannose as substrates, whereas the recently identified human GlcNAc kinase was specific to GlcNAc. Deletion of CaNAG2 or CaNAG5 markedly, and that of CaNAG1 moderately, attenuated the virulence of C. albicans in a mouse systemic infection model. Thus, it appears that GlcNAc metabolism of C. albicans is closely associated with its virulence.
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PMID:Identification and characterization of the genes for N-acetylglucosamine kinase and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida albicans. 1129 69


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