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)

Diethylpyrocarbonate (DEP) inhibits the catalytic activity of a cloned glutathione S-transferase from Schistosoma japonicum (Sj26GST) with a second-order rate constant of 474 M-1 min-1 at pH 7.0 and 25 degrees C. There is an accompanying increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. There was no evidence that tyrosine or cysteine residues were modified by DEP treatment nor did the enzyme undergo any major conformational change. Activity can be restored by treating the DEP-modified enzyme with hydroxylamine and the pH curve for inactivation indicates involvement of a residue with a pKa of 7.3. Complete inactivation of Sj26GST requires the modification of six histidine residues per subunit. Statistical analysis of residual enzyme activity versus number of groups modified showed that of the six modifiable groups, only one is critical for activity. Substrate protection suggests that this essential histidine residue is at or near the active site.
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PMID:Chemical modification of a cloned glutathione S-transferase from Schistosoma japonicum: evidence for an essential histidine residue. 775 42

The influence of different N-terminal affinity fusion domains on the product heterogeneity of recombinant proteins expressed in Escherichia coli was investigated. N-Terminal extended forms of the restriction endonuclease EcoRV with either glutathione-S-transferase [GST], histidine hexapeptide [(His)6], or a combination of GST and (His)6 [GST-(His)6] were compared to native EcoRV with respect to expression level, susceptibility to inclusion body formation and protein fragmentation. Fingerprinting of product heterogeneity was done by using two-dimensional (2-D) non-equilibrium pH-gradient electrophoresis with subsequent immunoblotting. Fusion proteins containing GST were poorly expressed compared to native EcoRV. In addition, GST fusion proteins were highly susceptible to in-vivo aggregation and fragmentation and displayed more heterogeneity on 2-D immunoblots. However, the sole presence of oligohistidine at the N-terminus of EcoRV proved to be advantageous. Fragmentation of (His)6-EcoRV was not observed and 2-D immunoblots did not show heterogeneous forms of the recombinant protein. In addition, fusion of the histidine-hexapeptide to the N-terminus of native EcoRV increased the expression level of the recombinant protein twofold compared to native EcoRV. Inclusion body formation of the (His)6-EcoRV fusion protein was intensive when cells were grown at 37 degrees C but not at 30 degrees C. The advantage of oligohistidine fusion to EcoRV was finally demonstrated by purifying soluble (His)6-EcoRV in a single-step procedure from crude cell lysates using immobilized metal chelate affinity chromatography.
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PMID:Comparison of N-terminal affinity fusion domains: effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoRV. 776 22

Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid residues, His84 and His85, that are suitably positioned for coordination to Ni(II)-IDA-agarose. This particular structural motif is lacking in GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineering afforded a mutant enzyme that displays affinity for Ni(II)-IDA-agarose, in contrast to the wild-type GST M1-1. The results identify a distinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins.
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PMID:Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes. 783 Dec 82

LIM homeodomain proteins are a family of recently characterized proteins which contain, in addition to a homeodomain, two tandem repeats of conserved Cys-His motifs termed as LIM domains. We have recently isolated several clones from a chinook salmon pituitary cDNA library that encode two novel LIM homeodomain proteins, Isl-2 and Isl-3, which are structurally related to rat Isl-1. In the present study, we used the salmon Isl-2 to determine the role of LIM domains in DNA binding. Several glutathione S-transferase (GST) fusion proteins containing either full length Isl-2 or various portions of this protein were expressed in bacteria. Zinc blot analysis reveals that the LIM domains produced in bacteria are capable of binding zinc. Gel shift analysis indicates that all homeodomain-containing fusion proteins are able to bind to a TAAT target sequence while the fusion proteins containing only the LIM domain are not. In contrast to a previous observation that the LIM domains of rat Isl-1 have an inhibitory role in DNA binding, full length salmon Isl-2 containing both the LIM domains and a homeodomain can bind to a TAAT target sequence. To further examine the role of LIM domains in DNA binding, several GST fusion proteins were used to select specific target DNA sequences from a pool of randomly incorporated oligonucleotides. Specific target DNAs were selected by fusion proteins containing the homeodomain or the full length Isl-2, but not by LIM domain only fusion proteins, indicating that the LIM domain alone is not involved in DNA binding. The selected target DNAs were cloned and sequenced. They revealed two classes of consensus, C/TTAATG/TG/A and C/TTAAGTG, for both the homeodomain and full length Isl-2. The two classes of consensus competed with each other for binding to the homeodomain. The equilibrium dissociation constants for DNA binding, estimated by Scatchard analysis, were similar for the homeodomain and full length Isl-2.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Zinc and DNA binding properties of a novel LIM homeodomain protein Isl-2. 799 75

We have constructed, expressed, and purified hexahistidine- and glutathione S-transferase (GST)-tagged Staphylococcal protein A. The histidine-tagged protein A bound efficiently to iminodiacetic acid (IDA)-Sepharose loaded with Zn2+, and the GST-protein A was efficiently retained by glutathione-Sepharose. Both recombinant forms of protein A can be used in the normal way to harvest immune complexes with IgG. Both forms of protein A can be released from the Sepharose matrix by mild procedures. The his6-protein A:antibody:antigen complexes can be released from the matrix with EDTA, and immunoprecipitates bound to GST-protein A can be released either by elution with glutathione or by digestion with thrombin. We tested this method with immunoprecipitates of the p40MO15 protein kinase, and found that they retained their ability to phosphorylate p33cdk2 after elution from the affinity matrices.
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PMID:Reversible immunoprecipitation using histidine- or glutathione S-transferase-tagged staphylococcal protein A. 805 65

Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAbSV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.
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PMID:Expression and purification of nonglycosylated SIV proteins, and their use in induction and detection of SIV-specific immune responses. 807 30

Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.
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PMID:Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. 823 Apr 41

Queuosine (Q) [7-(((4,5-cis-dihydroxy-2-cyclopenten-1-yl)amino)methyl)-7-deaz agu anosine] usually occurs in the first position of the anticodon of tRNAs specifying the amino acids asparagine, aspartate, histidine, and tyrosine. The hypermodified nucleoside is found in eubacteria and eucaryotes. Q is synthesized de novo exclusively in eubacteria; for eucaryotes the compound is a nutrient factor. In Escherichia coli the Q precursor (oQ), carrying a 2,3-epoxy-4,5-dihydroxycyclopentane ring, is formed from tRNA precursors containing 7-(aminomethyl)-7-deazaguanine (preQ1) by the queA gene product. A genomic queA mutant accumulating preQ1 tRNA was constructed. The QueA enzyme was overexpressed as a fusion protein with the glutathione S-transferase from Schistosoma japonicum and purified to homogeneity by affinity and anion-exchange chromatography. The enzyme QueA synthesizes oQ from preQ1 in a single S-adenosylmethionine- (AdoMet-) requiring step, indicating that the ribosyl moiety of AdoMet is transferred and isomerized to the epoxycyclopentane residue of oQ. The identity of oQ was verified by HPLC and directly combined HPLC/mass spectrometry. The formation of oQ was reconstituted in vitro, applying a synthetic RNA. A 17-nucleotide microhelix (corresponding to the anticodon stem and loop of tRNA(Tyr) from E. coli) is sufficient to act as the RNA substrate for oQ synthesis. We propose that QueA is an S-adenosylmethionine:tRNA ribosyltransferase-isomerase.
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PMID:A new function of S-adenosylmethionine: the ribosyl moiety of AdoMet is the precursor of the cyclopentenediol moiety of the tRNA wobble base queuine. 834 86

The human O6-methylguanine DNA methyltransferase (MGMT) repairs O6-methylguanine (O6-MG) in DNA at a much lower rate than the Escherichia coli Ada protein, and only MGMT repairs the altered base, O6-benzylguanine (O6-BG). The diversity in DNA repair properties between MGMT and Ada may be a result of divergent amino acid sequences outside their common proline-cysteine-histidine-arginine-valine (PCHRV) acceptor site. One notable sequence difference is an MGMT 28-amino acid carboxyl-terminal tail which is highly conserved among all mammalian alkyltransferases. The role of this tail sequence in substrate specificity was assessed by expressing full-length MGMT and Ada proteins, and mutant MGMT proteins lacking either 10 or 28 amino acids from the carboxyl terminus, as GST fusion proteins in alkyltransferase-deficient E. coli cells, and comparing rates of repair of O6-MG containing DNA and O6-BG by these fusion proteins at 4 degrees C and 37 degrees C. The MGMT carboxyl-terminal tail was not required for repair of O6-MG in DNA at 37 degrees C although the deletion of this tail sequence reversibly inhibited the ability of MGMT to repair O6-MG in DNA at 4 degrees C. Therefore, the absence of this region affects the ability of the protein to repair O6-MG in DNA at lower temperatures. Furthermore, removal of the tail sequence from MGMT decreased the rate of O6-BG repair 5-fold. We conclude that the 28-amino acid carboxyl-terminal MGMT tail, while not required for activity, modulates the rate of MGMT repair at reduced temperatures and plays a role in substrate specificity.
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PMID:The role of the carboxyl-terminal tail in human O6-methylguanine DNA methyltransferase substrate specificity and temperature sensitivity. 836 18

Each chick liver glutathione S-transferase CL 3 subunit contains three histidine residues: His142, His158 and His228. CL 3-3 can be inactivated by treating with diethylpyrocarbonate. The inactivation process is pH dependent and the pKa of the modified residue is 6.4. The second-order inhibition rate constant is 741 M-1min-1 at pH 7.0. Based on difference-spectrum and kinetic analysis, inactivation coincides with the modification of one histidine residue. However, hydroxylamine treatment of the diethylpyrocarbonate-modified enzyme only partially restored the activity (30-50%) of CL 3-3. By tryptic mapping and amino acid sequence analysis, His228 and Lys14 have been identified as the modified residues. Mutants with histidine to serine replacement (H142S and H158S) or C-terminal histidine deletion (des-H228) were constructed and over-expressed in Spodoptera frugiperda cells using a baculovirus system. The mutants are enzymically active. Furthermore, the des-H228 mutant can be inactivated by diethylpyrocarbonate. These results support the conclusion that histidines are not involved in the enzymic mechanism of CL 3-3.
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PMID:Site-directed mutagenesis and chemical modification of histidine residues on an alpha-class chick liver glutathione S-transferase CL 3-3. Histidines are not needed for the activity of the enzyme and diethylpyrocarbonate modifies both histidine and lysine residues. 843 37


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