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 775-amino-acid IE110 (or ICP0) phosphoprotein of herpes simplex virus (HSV) functions as an accessory transcription factor during the lytic cycle and plays a critical role in reactivation from latent infection. By immunofluorescence analysis, IE110 localizes in a novel pattern consisting of several dozen spherical punctate granules in the nuclei of DNA-transfected cells. We constructed a hybrid version of IE110 that contained an epitope-tagged domain from the N terminus of the HSV IE175 protein and lacked the IE110 N-terminal domain that confers punctate characteristics. This hybrid IE175(N)/IE110(C) protein gave an irregular nuclear diffuse pattern on its own but was redistributed very efficiently into spherical punctate granules after cotransfection with the wild-type HSV-1 IE110 protein. Similar colocalization interactions occurred with internally deleted forms of IE110 that lacked the zinc finger region or large segments from the center of the protein, including both cytoplasmic and elongated punctate forms, but C-terminal truncated versions of IE110 did not interact. In all such interactions, the punctate phenotype was dominant. Evidence that C-terminal segments of IE110 could also form stable mixed-subunit oligomers in vitro was obtained by coimmunoprecipitation of in vitro-translated IE110 polypeptides with different-size hemagglutinin epitope-tagged forms of the protein. This occurred only when the two forms were cotranslated, not when they were simply mixed together. An in vitro-synthesized IE110 C-terminal polypeptide also gave immunoprecipitable homodimers and heterodimers when two different-size forms were cross-linked with glutaraldehyde and reacted specifically with a bacterial glutathione S-transferase/IE110 C-terminal protein in far-Western blotting experiments. The use of various N-terminal and C-terminal truncated forms of IE110 in the in vivo assays revealed that the outer boundaries of the interaction domain mapped between codons 617 and 711, although inclusion of adjacent codons on either side increased the efficiency severalfold in some assays. We conclude that the C-terminal region of IE110 contains a high-affinity self-interaction domain that leads to stable dimer and higher-order complex formation both in DNA-transfected cells and in in vitro assays. This segment of IE110 is highly conserved between HSV-1 and HSV-2 and appears to have the potential to play an important role in the interaction with the IE175 protein, as well as in correct intracellular localization, but it is not present in the equivalent proteins from varicella-zoster virus, pseudorabies virus, or equine abortion virus.
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PMID:Identification of a dimerization domain in the C-terminal segment of the IE110 transactivator protein from herpes simplex virus. 815 88

Tomato cDNA and genomic clones were isolated by using as a probe a cDNA clone that had originally been identified by its ability to direct the synthesis of a biotin-containing polypeptide in Escherichia coli. The nucleotide sequences of the newly isolated cDNAs indicate that they are clones of a single mRNA molecule. However, one of the cDNA clones contains an insertion of a sequence which we identified as an unspliced intron. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed similarity to regions of previously sequenced biotin enzymes, indicating that the isolated cDNAs code for a biotin-containing protein. Portions of the cDNAs were expressed in E. coli as glutathione S-transferase or beta-galactosidase fusion proteins. Each fusion protein was purified and used to immunize rabbits. The resulting antisera recognized a 78-kDa biotin-containing polypeptide in tomato leaf extracts. In addition, both antisera specifically inhibited beta-methylcrotonyl-CoA carboxylase activity in extracts from tomato leaves. These characterizations have identified the isolated tomato cDNAs and genes as coding for the 78-kDa biotin subunit of beta-methylcrotonyl-CoA carboxylase. Comparison of the deduced amino acid sequence of the biotin subunit of beta-methylcrotonyl-CoA carboxylase with other biotin enzymes suggest that this subunit contains the biotin carboxylase and biotin carboxyl-carrier domains.
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PMID:Molecular cloning of cDNAs and genes coding for beta-methylcrotonyl-CoA carboxylase of tomato. 816 72

Escherichia coli cells were transformed with an expression vector constructed by inserting a DNA fragment encoding a Kazal-type trypsin inhibitor from mouse seminal vesicle into pGEX-2. The cloned cells were able to produce a high yield of a chimeric polypeptide made by fusing the trypsin inhibitor to glutathione S-transferase. The chimeric polypeptide could be purified through an affinity column of glutathione agarose beads. The purified protein could be digested with thrombin to release the recombinant trypsin inhibitor which could be further purified by HPLC of the thrombin digests on a reverse-phase C4 column. The recombinant trypsin inhibitor was homogeneous and showed trypsin inhibitor activity as strong as that of the naturally occurring trypsin inhibitor.
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PMID:Purification and biochemical characterization of a recombinant mouse seminal vesicle trypsin inhibitor produced in Escherichia coli. 816 70

Dimethyl sulfoxide-differentiated U937 (dU937) cells express high affinity G-protein-coupled receptors for leukotriene (LT)D4 and LTB4 and, as described here, specific binding sites for LTC4. The specific binding of [3H]LTC4 was of low affinity (KD = 26 nM) and high abundance (Bmax = 33 pmol/mg of protein), as compared to LTD4 and LTB4 receptors. In addition, although [3H]LTC4 specific binding was enhanced by divalent cations, it was not inhibited by nonhydrolyzable GTP analogs. [3H]LTC4 specific binding to dU937 cell membranes does not have, therefore, the characteristics of binding to a G-protein-coupled receptor. Competition for [3H]LTC4 specific binding to dU937 cell membranes by leukotrienes and related analogs, including N-methylated LTC4, as well as glutathione, suggested a dependence on the presence of an arachidonic acid backbone, although varying degrees of saturation were well tolerated, and that the glutathione moiety of LTC4 in particular was important in determining affinity. The possibility that [3H]LTC4 specific binding was to a member of the glutathione S-transferase (GST) family of enzymes, such as LTC4 synthase, cytosolic GST, or microsomal GST, was therefore investigated. [3H]LTC4 specific binding sites could be separated from LTC4 synthase and cytosolic GSTs by differential detergent solubilization, but cofractionated with microsomal GST during solubilization and subsequent anion exchange chromatography. In membranes that were depleted of LTC4 synthase and cytosolic GSTs, 125I-azido-LTC4 (a photoaffinity probe based on LTC4) specifically photolabeled in a cation-dependent manner a 17-kDa polypeptide that was comparable in mass to the microsomal GST polypeptide. Furthermore, [3H]LTC4 bound specifically to purified human microsomal GST with the same characteristics as to the endogenous dU937-cell membrane specific binding sites. The principal [3H]LTC4 specific binding site present in dU937 cells, therefore, is not a G-protein-coupled receptor, LTC4 synthase, or cytosolic GSTs, but is microsomal GST. Finally, the 1:3 stoichiometry of [3H]LTC4 specific binding to purified microsomal GST is consistent with the enzyme functioning as a homotrimer.
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PMID:Microsomal glutathione S-transferase is the predominant leukotriene C4 binding site in cellular membranes. 817 95

Marek's disease virus (MDV) is an oncogenic avian herpesvirus whose genomic structure is similar to those of herpes simplex virus and varicella-zoster virus. Repeat regions of the MDV genome have been intensively investigated because of a potential relationship to MDV oncogenicity and abundant expression of immediate-early transcripts. In this study, a 1.6-kb immediate-early transcript was localized to the BamHI-I2 region by Northern (RNA) hybridization analysis. With cDNA cloning and sequencing, two cDNAs of 1.4 kb (C1) and 1.35 kb (C2) were identified. Both cDNAs are derived from spliced mRNAs spanning the BamHI-H and -I2 fragments. C1 and C2 use the same splice acceptors and 3' ends, but they differ at their 5' ends and utilize different splice donors. The upstream promoter-enhancer region of C1 cDNA has been defined as a bidirectional regulatory region shared by the MDV pp38 gene. Sequencing analysis shows two small open reading frames (ORFs) within each cDNA (ORF1a and ORF2 in C1, ORF1b and ORF2 in C2). Potential ORFs of the sequence have no significant homology with any known protein in the Swiss-Protein data base. DNA fragments encoding ORF1a and ORF1b were cloned into pGEX-3X vectors to produce glutathione S-transferase fusion proteins and induce antisera. In Western blot (immunoblot) analysis of MDV-infected-cell lysates, a 14-kDa polypeptide was identified by antisera against both ORF1a and ORF1b. This 14-kDa protein is expressed in cells which are lytically infected with MDV strains GA, Md11 passage 14 (oncogenic), and Md11 passage 83 (attenuated), as well as in the latently MDV-infected and transformed MSB-1 cell line.
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PMID:Identification of an immediate-early gene in the Marek's disease virus long internal repeat region which encodes a unique 14-kilodalton polypeptide. 818 98

Transcription of a putative mitochondrial gene (orf138) has previously been correlated with Ogura cytoplasmic male-sterility (CMS) in rapeseed cybrids. In this paper, studies performed on a Brassica cybrid with a different organization of the orf138 locus confirm this association. We also show that mitochondria isolated from male-sterile rapeseed plants synthesize a polypeptide of 19 kDa, which is absent in fertile revertants. Antibodies against a glutathione S-transferase-ORF138 fusion protein were raised to establish that this 19 kDa polypeptide is the product of orf138. The anti-ORF138 serum was used to demonstrate that the orf138 translation product occurs only in sterile cybrids and co-purifies with the mitochondrial membrane fraction.
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PMID:Ogura cytoplasmic male-sterility (CMS)-associated orf138 is translated into a mitochondrial membrane polypeptide in male-sterile Brassica cybrids. 820 45

Microsomal glutathione S-transferase was labeled by the fluorescence probe N-(1-pyrenyl)maleimide which modified 1 mol thiol residue/mol protein. The enzyme activity increased about tenfold after the binding. The pyrene-labeled microsomal glutathione S-transferase exhibited two fluorescence bands which are typical of pyrene; one at 393 nm attributable to unassociated pyrenes, the other at 480 nm attributable to pyrene excimers (excited dimers). The excimeric fluorescence increased at high protein concentrations indicating a shift of the equilibrium of labeled polypeptide chains from trimeric complexes, the functional unit of microsomal glutathione S-transferase, to larger aggregates. At 25 degrees C and at a 1% Triton X-100 concentration, the calculated equilibrium constant of this process is 65 microM. Along with the formation of large aggregates, a progressive increase of the enzymic activity was observed. Thus, N-(1-pyrenyl)maleimide appears to be a very useful probe to study the supramolecular structure of this enzyme.
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PMID:Aggregation of pyrene-labeled microsomal glutathione S-transferase. Effect of concentration. 822 8

Two clones coding for class-alpha glutathione S-transferase were isolated from a chicken liver cDNA library. The full-length clone (933 bp) encodes a polypeptide comprising 221 amino acids with a molecular mass of 25,298 Da, including the initiator methionine. The partial clone (935 bp) encodes the C-terminal 193 amino acids of a different class-alpha glutathione S-transferase. The deduced primary amino acid sequence of the full-length clone has a 66% identical sequence with other class-alpha glutathione S-transferases.
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PMID:Nucleotide sequence of class-alpha glutathione S-transferases from chicken liver. 824 Dec 81

Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.
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PMID:Identification and sequence analysis of cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate. 824 4

Recombinant hsc70, a purified glutathione S-transferase (GST) fusion protein containing the C-terminal domain of hsc70 (GST-Ct), and an internal 18-kDa polypeptide located immediately after the 44-kDa ATPase domain of hsc70 were investigated for their peptide binding properties. The dissociation constants of the S-peptide for native hsc70 (Kd = 5-8 microM), GST-Ct (Kd = 6.5 microM), and the 18-kDa fragment (Kd = 8.1 microM) are virtually identical. In addition, polylysine and (Pro-Pro-Gly)5 do not show high affinity toward hsc70, GST-Ct, and the 18-kDa fragment, whereas peptide GT4 and P3a show comparably high affinity toward these polypeptides. These observations indicate that the peptide binding domain of hsc70 is confined in the internal 18-kDa fragment.
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PMID:Identification of the peptide binding domain of hsc70. 18-Kilodalton fragment located immediately after ATPase domain is sufficient for high affinity binding. 825 14


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