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)

c-Mil is the avian homologue of the mammalian serine/threonine kinase c-Raf-1. c-Mil/Raf is a mediator of signal transduction leading to gene expression via the c-Jun DNA-binding site, AP-1. Here we show that c-Mil immunopurified from MC29-virus-transformed quail fibroblasts phosphorylates c-Jun in vitro near its N terminus (Ser-63 and -73). Furthermore, the viral oncogene product Gag-Mil of the avian wild-type retrovirus MH2 phosphorylates c-Jun in vitro. A contribution by other known kinases phosphorylating c-Jun, such as the mitogen-activated protein kinases (MAPKs) and the c-Jun N-terminal kinases, was excluded by control reactions. c-Raf-1 and c-Jun directly interact in vitro as shown by various immobilized glutathione S-transferase-Raf fusion proteins which specify the cysteine-rich region of c-Mil/Raf as the major N-terminal binding site. An additional minor binding site is located in the C-terminal region. The biological relevance of these results is demonstrated by coimmunoprecipitation of c-Jun and c-Mil from 32P-labeled MC29- and MH2-transformed fibroblasts as well as normal quail embryo fibroblasts, whereby c-Jun was identified by tryptic phosphopeptide analysis. The complexed c-Jun exhibits a decreased electrophoretic mobility corresponding to a more highly phosphorylated state. Cell fractionation analyses indicate that the c-Mil/c-Jun complex is located in the cytoplasm. The data demonstrate that c-Jun can be a direct target of the protein kinase c-Mil/Raf, suggesting an alternative pathway, which leads to c-Jun phosphorylation independent of the MAPKs and MAPK-related proteins.
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PMID:Direct interaction and N-terminal phosphorylation of c-Jun by c-Mil/Raf. 787 94

Rhodostomin (Rho) from snake venom, a potent inhibitor of platelet aggregation, contains 68 amino acids having an RGD sequence and 12 cysteine residues. A chemically synthesized Rho gene was cloned and expressed in Escherichia coli. The expression of Rho gene fused with the glutathione S-transferase (GST) gene was about 10-30% of total cell proteins. The Rho-fusion protein could be recognized by antibodies raised against either a native Rho peptide or a synthetic peptide. The purified GST-Rho coated on culture plates facilitated the attachment of human hepatoma cells, which was inhibitable by co-incubation with a synthetic hexapeptide GRGDSP but not with a related peptide of GRGESP, suggesting that the E. coli-expressed Rho-fusion protein was properly folded and biologically functional.
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PMID:Rhodostomin, an RGD-containing peptide expressed from a synthetic gene in Escherichia coli, facilitates the attachment of human hepatoma cells. 791 92

The murine aromatic hydrocarbon ([Ah]) gene battery consists of at least six genes that code for two functionalizing (Phase I) enzymes and four non-functionalizing (Phase II) enzymes. These enzymes are induced by compounds such as aromatic hydrocarbons and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that bind to the cytosolic Ah receptor protein. Studies in rodents indicate that certain enzymes of this battery, namely cytochrome P4501A1 (CYP1A1), UDP-glucuronosyltransferase (UGT1*06) and NAD(P)H: quinone acceptor oxidoreductase (NMO1) are induced by the synthetic antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII). The induction of [Ah] gene battery enzymes and the levels of reduced glutathione (GSH) were examined in mouse Hepa-1c1c7 hepatoma wild-type cells (wt), a CYP1A1 metabolism-deficient mutant (c37) and an Ah receptor nuclear translocation-defective mutant (c4). DHII and TCDD increased the activities of ethoxyresorufin O-deethylase, an indicator of CYP1A1 activity, as well as NMO1, UGT1*06, cytosolic aldehyde dehydrogenase class 3 and glutathione S-transferase form A1 in wt cells, but had little or no induction effect in c37 or c4 cells. DHII and TCDD differed in their effects on GSH levels; while DHII increased GSH levels 3-fold in wt, but not at all in c37 or c4 cells, TCDD had no effect on GSH levels in any cell type. However, GSH levels were enhanced in both wt and c4 cells by tert-butyl hydroquinone (TBHQ). L-Buthionine S,R-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, prevented DHII-induced increases in wt cell GSH. The increase in GSH levels occurred after 8 h, while the induction of enzymes occurred within 4 h. The induction of the higher GSH levels in wt cells by DHII and TBHQ correlated with increases in intracellular levels of the GSH precursor thiol cysteine, as well as with increased activities of gamma-glutamylcysteine synthetase, the rate-limiting enzyme of GSH synthesis. However, TBHQ-mediated GSH increases in c4 cells were accompanied by increased gamma-glutamylcysteine synthetase activity with no change in intracellular cysteine concentration. The results suggest that DHII induction of [Ah] gene battery enzymes requires a functional Ah receptor, but not the functional gene product CYP1A1. Furthermore, metabolism, possibly via CYP1A1, appears to be required for DHII to enhance intracellular levels of cysteine and GCS activity that result in higher GSH levels.
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PMID:Regulation of [Ah] gene battery enzymes and glutathione levels by 5,10-dihydroindeno[1,2-b]indole in mouse hepatoma cell lines. 795 76

The regulatory domain of protein kinase C gamma (PKC gamma) contains the following functional elements which can interact with lipids: the pseudosubstrate motif within the first variable region (V1), cysteine-rich domains, Cys1 and Cys2 which contain zinc and bind phorbol dibutyrate (PDBu)/diacylglycerol, and the calcium-dependent lipid binding domain (CaLB). The function of individual or combined segments of the regulatory domain was investigated, using glutathione S-transferase (GST) fusion proteins and mixed micellar or liposomal assays. GST-Cys1 and GST-Cys2 bound PDBu with comparable affinity (Kd = 14-17 nM). GST-Cys1Cys2 yielded a protein with a PDBu binding affinity of 3.4 nM, in the presence of calcium, similar to that of intact PKC gamma (Kd = 2.6 nM). The phosphatidylserine (PS) dependence of PDBu binding was highly cooperative for all fusion proteins tested with Hill numbers (n) lying in the range of 3.5-4.8, similar to values obtained for intact PKC gamma. While Hill numbers were similar under all conditions, the PS concentration necessary for half-maximal PDBu binding was dependent upon the nature and presence of divalent cations. The PS requirement was lowest in the presence of calcium for GST-Cys1, GST-Cys2, and GST-Cys1Cys2 (Km for PS = 11, 14, and 12 mol %, respectively) but still significantly above the value for intact PKC gamma (5.4 mol %). The data establish Cys1 and Cys2 as independent PDBu binding domains that are modulated by divalent cations. While PDBu binding affinity to a GST-V1Cys1 fusion protein (Kd = 36 nM) was comparable to that of GST-Cys1, the CaLB domain dramatically reduced PDBu binding affinity of GST-Cys2CaLB (Kd = 912 nM). This effect of the CaLB domain on PDBu binding to Cys2 suggests that PDBu/diacylglycerol binding to native PKC gamma may occur at Cys1 and that the Cys2 domain may serve another regulatory function.
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PMID:The regulatory region of protein kinase C gamma. Studies of phorbol ester binding to individual and combined functional segments expressed as glutathione S-transferase fusion proteins indicate a complex mechanism of regulation by phospholipids, phorbol esters, and divalent cations. 805 Oct 84

The vaccinia virus-encoded protein VP39 is a poly(A) polymerase subunit that stimulates the formation of long poly(A) tails as well as a cap-specific mRNA (nucleoside-2'-O-)-methyltransferase. We have carried out mutagenesis studies aimed at locating regions of VP39 which are important for these activities. The open reading frame encoding VP39 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. The affinity-purified protein had both mRNA modification activities, before and after removal of the glutathione S-transferase domain. Truncation, charge cluster-->Ala scanning, and Cys-->Ser substitution mutations of VP39 were made, and the proteins were synthesized, purified, and analyzed. Deletion of the RNA binding domain, experimentally localized within the carboxyl-terminal 112 amino acids, resulted in the loss of both mRNA modification activities. Eleven of the 21 charge cluster-->Ala mutated proteins had low to nondetectable methyltransferase activity. Four of those 11 also lacked adenylyl-transferase stimulatory function, whereas the remainder had amino acid substitutions that selectively affected methyltransferase activity. However, no mutated proteins lacking adenylyltransferase stimulatory function but possessing methyltransferase activity were isolated by the procedures used. Neither of the 2 cysteine residues in VP39 was necessary for either mRNA modification activity.
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PMID:Mutational analysis of a multifunctional protein, with mRNA 5' cap-specific (nucleoside-2'-O-)-methyltransferase and 3'-adenylyltransferase stimulatory activities, encoded by vaccinia virus. 805 Nov 70

Polyclonal antibodies were raised to partial and full-length synthetic peptides of human papillomavirus type 16 (HPV-16) E5. Antisera specificity for HPV-16 E5 was demonstrated by their ability to recognize not only their peptide immunogens but also full-length peptide and a glutathione S-transferase-E5 fusion protein. The most reactive antiserum, PE-6, raised to a full-length peptide, was used in Western blot analysis to identify HPV-16 E5 protein from exfoliated cervical cells. A strong, single band at approximately 20K was detected in two of six HPV-16-positive samples from women with a history of low-grade cervical intraepithelial neoplasia. The apparent M(r) by SDS-PAGE suggests that HPV-16 E5 forms homodimers in vivo, but not through cysteine linkage.
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PMID:Detection of E5 oncoprotein in human papillomavirus type 16-positive cervical scrapes using antibodies raised to synthetic peptides. 807 47

Different domains of the serine/threonine kinase, raf-1, were expressed as fusion proteins with glutathione S-transferase (GST) in Escherichia coli and purified to near homogeneity by affinity chromatography. A cysteine-rich domain of raf-1 was found to contain 2 mol of zinc (molar basis), similar to analogous cysteine-rich domains of protein kinase C. GST-fusion proteins, containing the cysteine-rich domain of raf-1, bound to liposomes in a phosphatidylserine-dependent manner. In contrast to protein kinase C, the translocation of raf-1 was not dependent upon diacylglycerol, phorbol ester, or calcium, nor did raf-1 bind phorbol esters. A GST-fusion protein encoding residues 1-147 of raf-1 bound to normal GTP-ras with high affinity, but not to mutant GTP-Ala35 ras; no binding was detected to GDP-ras. The binding of a smaller fusion protein (residues 1-130 of raf-1) was about 10-fold weaker, inferring that a 17-amino acid sequence represents a critical binding determinant in intact raf-1. These residues are adjacent to the amino-terminal end of, and partially extend into, the cysteine-rich domain (amino acids 139-184). A synthetic peptide corresponding to this 17-amino acid sequence blocked the interaction of raf-1 with ras. The function of the cysteine-rich region of raf-1 homologous to protein kinase C is to promote translocation of raf-1 kinase to membranes and to form part of the high affinity binding site for GTP-ras.
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PMID:The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras. 814 97

The interaction of ebselen(2-phenyl-1,2-benzisoselenazol-3(2H)-one) with rat liver cytosolic glutathione S-transferases (GSTs) and the plant cysteine protease, papain, was studied as cysteine residues are important for the activity of these enzymes. The capacity of GST 1-2 and 3-4 for ebselen binding is similar (1.5 mol ebselen/mol GST isozyme), while GST 2-2 and GST 7-7 bind 0.3 and more than 2.0 mol ebselen/mol GST isozyme, respectively. Ebselen does not bind to N-ethylmaleimide-treated GST, and its binding to GST is prevented by 5 mM thiols. Ebselen irreversibly inactivates the different GST isozymes with a second order rate constant ranging from 20 to 2250 M-1 sec-1 for the different subunits. GST inhibition by ebselen is partially restored by 5 mM thiols. Ebselen binds to untreated papain and to cysteine-treated papain at a ratio of about 0.1 and 0.75 mol ebselen/mol papain, respectively. Ebselen does not bind to N-ethylmaleimide-treated papain, and its binding to papain is interfered with by added thiols. Papain is inactivated by ebselen with a second order rate constant of 1800 M-1 sec-1 in the absence of thiols. However, in the presence of GSH, 2-mercaptoethanol or sodium borohydride, ebselen exerts an activating effect on papain. The binding of ebselen by a seleno-sulfide bond to cysteine residues of GSTs and papain leads to their inactivation.
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PMID:Interaction of ebselen with glutathione S-transferase and papain in vitro. 814 99

This manuscript describes changes in plasma lipid profiles and parameters of oxidative status in the plasma and liver of rats fed 5 different fatty acids: 95% eicosapentaenoic acid, 92% docosahexaenoic acid (DHA), corn oil (n-6), 1-mono-(carboxymethylthio)-tetradecane (CMTTD) and palmitic acid (controls) for 3 months. At the given doses both EPA and the 3-thia fatty acid, CMTTD, caused a significant decrease in plasma triglycerides, phospholipids, free fatty acids and cholesterol. DHA decreased plasma free fatty acids and cholesterol, while corn oil feeding reduced only plasma free fatty acids. Plasma and hepatic vitamin E levels were significantly decreased in EPA, DHA and CMTTD fed rats, but remained unchanged in corn oil fed rats. Plasma glutathione was noted to decrease after EPA and DHA feeding but remained unchanged in other groups. However, hepatic glutathione content was increased in EPA, DHA and CMTTD fed rats, whereas cysteine levels were noted to decrease. As hepatic levels of cysteinylglycine remained unchanged, increased rate of cellular glutathione synthesis rather than its decreased degradation is likely to contribute to the increased hepatic glutathione content in EPA, DHA and CMTTD fed rats. Except for reduction in the levels of plasma lipid peroxidation caused by CMTTD, no significant changes were noted between the different treatment groups. Hepatic lipid peroxidation was elevated only in rats given DHA. Furthermore, our results show that EPA and DHA cause minimal imbalance of the peroxisomal H2O2 metabolising enzymes as compared to CMTTD. In addition, contrary to the potent peroxisome proliferator compound CMTTD which decreased the activities of glutathione transferase and glutathione peroxidase, EPA and DHA increased the activities of these detoxification enzymes.
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PMID:Modulation of plasma and hepatic oxidative status and changes in plasma lipid profile by n-3 (EPA and DHA), n-6 (corn oil) and a 3-thia fatty acid in rats. 816 62

Rat liver glutathione S-transferase 3-3 (GST, EC 2.5.1.18), a triple mutant with all three cysteine residues replaced with serine (CallS) and a quadruple mutant with a Tyr-115 to phenylalanine substitution on CallS (CallSY115F) were overexpressed in Escherichia coli under the control of a phoA promoter. Using this system, we obtained over 35 mg of fully active pure protein/litre of cell medium. GST 3-3 and CallS mutant were modified with 1-chloro-2,4-dinitrobenzene (CDNB), a model substrate for the enzyme, in the absence of GSH. Dinitrophenol, but not S-methylglutathione, inhibits this process. The dinitrophenyl groups are readily removed from the enzyme with GSH, but much more slowly with dithiothreitol. Results from peptide mapping and amino acid sequence analyses indicate that CDNB modifies the cysteine residues and Tyr-115 on wild-type GST 3-3, but only Tyr-115 on CallS. In addition, CDNB cannot modify the CallSY115F mutant. We propose that Tyr-115 is located at or near the H-site of GST 3-3.
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PMID:Reversible modification of rat liver glutathione S-transferase 3-3 with 1-chloro-2,4-dinitrobenzene: specific labelling of Tyr-115. 825 Aug 42


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