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)

Epithelial liver cells of the Chinese hamster (CHEL cells) were propagated in culture for 35 passages. At favourable cell densities, the population doubling time in normal medium, was 20 h. L-Tyrosine amino transferase activity was retained at a measurable level, but its enhancement by dexamethasone was detected solely in cells of early passages. Pyruvate kinase was strongly activated by fructose-1,6-biphosphate at low substrate concentrations. These enzymatic properties suggest that the CHEL cells are derived from a sub-population of parenchymal hepatocytes or from cells closely related to parenchymal hepatocytes. With a lag period of a few hours, CHEL cultures metabolized benzo[a]pyrene. In cell homogenates the various monooxygenase activities investigated were below the detection limits. However, other xenobiotic-metabolizing activities, such as cytochrome P-450 reductase, glutathione transferase and UDP-glucuronosyl-transferase were high, with levels comparable to those observed in freshly isolated rat parenchymal cells. Epoxide hydrolase activity was also detected, but was lower than in the liver. The CHEL cells were able to activate benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and aflatoxin B1 to mutagens, as shown in a co-culture assay with V79 cells, in which acquisition of resistance to 6-thioguanine was studied. At early passages, the CHEL cells had a near diploid set of chromosomes. Then, gradually the frequency of cells with slight changes in the number of chromosomes and the frequency of tetraploids were increased. During the observation period (up to passage 20) the modal number of chromosomes shifted from 22 to 23. No gross morphological changes in the cultures were noticed during the 20 passages.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of an epithelial, nearly diploid liver cell strain, from Chinese hamster, able to activate promutagens. 290 Oct 26

We have isolated the murine Limk1 gene, which is a single copy gene located at the distal end of mouse chromosome 5. Limk1 exhibits a 95% homology to the human homologue, LIMK, which contains two LIM domains and a putative protein kinase domain. Although Limk1 and LIMK contain all motifs found in catalytic kinase domains, amino acids previously described to be diagnostic of either serine/threonine- or tyrosine-kinases are not present. It is demonstrated that GST-Limk1-fusion protein can autophosphorylate on serine, tyrosine and threonine residues in vitro and that mutation of residue D460 within the IHRDL motif abolishes kinase activity. Northern blot showed preferential expression of a 3.5 kb message in adult spinal cord and brain. In situ hybridisation confirmed high expression levels in the nervous system, particularly in the spinal cord and the cranial nerve and dorsal root ganglia. Limk1 also contains two tandem LIM-domains. These zinc-finger like domains can mediate protein-protein interactions and have been described in nuclear and cytoskeletal proteins. The combination of LIM- and kinase domains may provide a novel route by which intracellular signalling can be integrated.
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PMID:Limk1 is predominantly expressed in neural tissues and phosphorylates serine, threonine and tyrosine residues in vitro. 747 47

Activation of human platelets by cross-linking of the platelet low-affinity IgG receptor, the Fc gamma receptor IIA (Fc gamma-RIIA), or by collagen is associated with rapid phosphorylation on tyrosine of the non-receptor tyrosine kinase syk. Phosphorylation is still observed, albeit sometimes reduced, in the presence of a combination of a protein kinase C inhibitor, Ro 31-8220, and the intracellular calcium chelator, BAPTA-AM, demonstrating independence from phosphoinositide-specific phospholipase C (PLC) activity. In contrast, the combination of Ro 31-8220 and BAPTA-AM completely inhibits phosphorylation of syk in thrombin-stimulated platelets. Phosphorylation of syk increases its autophosphorylation activity measured in a kinase assay performed on syk immunoprecipitates. Fc gamma-RIIA also undergoes phosphorylation in syk immunoprecipitates from platelets activated by cross-linking of Fc gamma-RIIA but not by collagen, suggesting that it associates with the kinase. Consistent with this, tyrosine-phosphorylated Fc gamma-RIIA is precipitated by a glutathione S-transferase (GST) fusion protein containing the tandem src homology (SH2) domains of syk from Fc gamma-RIIA- but not collagen-activated cells. Two uncharacterized tyrosine-phosphorylated proteins of 40 and 65 kDa are uniquely precipitated by a GST fusion protein containing the tandem syk-SH2 domains in collagen-stimulated platelets. A peptide based on the antigen recognition activation motif (ARAM) of Fc gamma-RIIA, and phosphorylated on the two tyrosine residues found within this region, selectively binds syk from lysates of resting platelets; this interaction is not seen with a non-phosphorylated peptide. Kinase assays on Fc gamma-RIIA immunoprecipitates reveal the constitutive association of an unidentified kinase activity in resting cells which phosphorylates a 67 kDa protein. Syk is not detected in Fc gamma-RIIA immunoprecipitates from resting cells but associates with the receptor following activation and, together with Fc gamma-RIIA, is phosphorylated in the kinase assay in vitro. These results demonstrate that syk is activated by Fc gamma-RIIA cross-linking and collagen, independent of PLC, suggesting that it may have an important role in the early events associated with platelet activation. The association of syk with Fc gamma-RIIA appears to be mediated through the tandem SH2 domains in syk and the ARAM motif of Fc gamma-RIIA. A similar interaction may underlie the response to collagen, suggesting that its signalling receptor contains an ARAM motif.
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PMID:Syk interacts with tyrosine-phosphorylated proteins in human platelets activated by collagen and cross-linking of the Fc gamma-IIA receptor. 748 83

The gene encoding the C-terminal protease domain (27 kDa) of the nuclear inclusion protein a of turnip mosaic potyvirus C5 was cloned and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. Two forms of the protease (27 and 25 kDa) were purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography and exhibited the specific proteolytic activity when a synthetic undecapeptide, Glu-Pro-Thr-Val-Tyr-His-Gln-Thr-Leu-Asn-Glu, or an in vitro translation product of the polyprotein containing the cleavage site between the nuclear inclusion protein b and the capsid protein, was used as a substrate. The purified proteases showed a Km of 1.15 +/- 0.16 mM and a Vmax of 0.74 +/- 0.091 mumol/mg/min with the synthetic peptide substrate. The 25-kDa protein was found to be generated by the cleavage between Ser223 and Gly224 near the C-terminus of the 27-kDa protease and to retain the specific proteolytic activity. The point mutation of Asp81 or Cys151, two putative active site residues in the 27-kDa protease, to Asn or Ser, respectively, prevented the generation of the 25-kDa protein and diminished the proteolytic activity of the protease drastically, suggesting that the 27-kDa protease cleaves itself between Ser223 and Gly224 to generate the 25-kDa protein.
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PMID:Expression, purification, and identification of a novel self-cleavage site of the Nla C-terminal 27-kDa protease of turnip mosaic potyvirus C5. 749 76

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary neutrophils from patients with CML, the major novel tyrosine-phosphorylated protein is CRKL, an SH2-SH3-SH3 linker protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene product. Anti-CRKL immunoprecipitates from CML cells, but not normal cells, were found to contain p210BCR/ABL and c-ABL. Several other phosphoproteins were also detected in anti-CRKL immunoprecipitates, one of which has been identified as paxillin, a 68-kDa focal adhesion protein which we have previously shown to be phosphorylated by p210BCR/ABL. Using GST-CRKL fusion proteins, the SH3 domains of CRKL were found to bind c-ABL and p210BCR/ABL, while the SH2 domain of CRKL bound to paxillin, suggesting that CRKL could physically link p210BCR/ABL to paxillin. Paxillin contains three tyrosines in Tyr-X-X-Pro (Y-X-X-P) motifs consistent with amino acid sequences predicted to be optimal for binding to the CRKL-SH2 domain (at positions Tyr-31, Tyr-118, and Tyr-181). Each of these tyrosine residues was mutated to a phenylalanine residue, and in vitro binding assays indicated that paxillin tyrosines at positions 31 and 118, but not 181, are likely to be involved in CRKL-SH2 binding. These results suggest that the p210BCR/ABL oncogene may be physically linked to the focal adhesion-associated protein paxillin in hematopoietic cells by CRKL. This interaction could contribute to the known adhesive defects of CML cells.
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PMID:CRKL links p210BCR/ABL with paxillin in chronic myelogenous leukemia cells. 749 40

A gene named stp1+, coding for a 17.5-kDa protein, that rescues cdc25-22 when overexpressed, has been previously isolated from fission yeast. Here we describe the expression and purification of Stp1 protein as a fusion with the glutathione S-transferase in E. coli and its kinetic characterisation. Stp1 deduced protein sequence shows an high homology to members of a class of cytosolic low M(r) protein phosphatase previously known to exist only in mammalian species. Stp1 has a kinetic behaviour that appears to be intermediate with respect to the two isoenzymatic forms of low M(r) protein tyrosine phosphatases present in mammalian tissues. These differing kinetic characteristics are mainly due to the sequence 45-56 that is spatially close to the active site pocket.
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PMID:Expression, purification and kinetic behaviour of fission yeast low M(r) protein-tyrosine phosphatase. 749 7

Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21ras or injection of oncogenic p21ras has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21ras/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway.
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PMID:Insulin-stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway. 749 78

Immunoprecipitates of metabolically labeled PC12 cells consistently contained a 43-kDa protein that was associated with Shc, a signal-transducing protein with a single SH2 domain. Following affinity chromatography with immobilized recombinant glutathione S-transferase (GST)-Shc fusion protein, the 43-kDa protein was identified as actin by mass spectrometry and immunoblotting. Cosedimentation experiments using purified actin and GST-Shc showed that Shc binds directly to F-actin, confirming Shc-actin interaction in vivo. Various GST-truncated Shc fusion proteins were prepared and used in actin cosedimentation assays. Constructs containing the SH2 and collagen homology domains were not precipitated, and those containing the amino-terminal domain were. Thus, Shc-actin interactions do not occur in the region of tyrosine phosphorylation and leave the SH2 domain free to bind to other tyrosine-phosphorylated molecules. Although the major pool of Shc in unstimulated PC12 cells is soluble, two other pools are associated with the cytoskeleton and the submembranous cytoskeleton. Upon nerve growth factor stimulation, approximately 50% of the soluble Shc translocates to both cytoskeleton environments within 2 min, decreasing thereafter. When cells were pretreated with cytochalasin D, a drug that disrupts actin filaments, Shc translocation to the cytoskeleton was abolished. However, in the submembranous fraction, the Shc level was elevated in resting cells following cytochalasin D treatment. The kinetics of translocation, compared to mitogen-activated protein kinase activation, and the nature of the Shc-actin interaction suggest that the cytoskeletal association of Shc, induced by growth factors, may be related to membrane ruffling and actin fiber reorganization.
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PMID:Src homologous and collagen (Shc) protein binds to F-actin and translocates to the cytoskeleton upon nerve growth factor stimulation in PC12 cells. 749 22

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.
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PMID:T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T). 750 57

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.
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PMID:Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides. 750 71


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