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 cDNA encoding the C-terminal nucleotide-binding domain (NBD2) from mouse P-glycoprotein involved in multidrug resistance was obtained from adrenal cell mRNA and amplified by reverse transcriptase polymerase chain reaction. NBD2 was highly overexpressed in Escherichia coli in fusion with glutathione S-transferase and could be purified after efficient thrombin cleavage. Both fused and purified NBD2 bound TNP (2',3'-O-(2,4,6-trinitrophenyl))- derivatives of nucleotides with high affinity. TNP-ATP or TNP-ADP binding at micromolar concentrations produced a characteristic blue-shifted enhancement of extrinsic fluorescence and was specifically prevented or chased by ATP or ADP at millimolar concentrations. A similar affinity binding was monitored by quenching of intrinsic fluorescence. The spectrum of fusion protein, containing 5 tryptophan residues, was maximally quenched at 328 nm upon interaction with TNP-nucleotides. TNP-GTP exhibited a lower affinity than TNP-ATP but produced a higher maximal quenching (44% instead of 28%). The intrinsic fluorescence of purified NBD2, containing a single tryptophan residue, exhibited a narrow spectrum with a maximum at 328 nm characteristic of a hydrophobic tryptophan environment. A high quenching was observed upon nucleotide interaction with similar affinity. The results put forward a functional role for the tryptophan-containing sequence of P-glycoprotein NBD2 that was not detected up to now.
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PMID:Overexpression and purification of the carboxyl-terminal nucleotide-binding domain from mouse P-glycoprotein. Strategic location of a tryptophan residue. 791 13

We previously reported the isolation from Entamoeba histolytica of a novel rac family protein kinase gene, termed Ehrac1, for "related to cAMP-dependent protein kinases and protein kinase Cs". To study the function and properties of this kinase gene further, we fused the full-length coding region and the truncated catalytic domain of the Ehrac1 gene in frame with the gene encoding glutathione S-transferase in the pGEX-KG vector and expressed the fusion in Escherichia coli. The thrombin-cleaved and uncleaved fusion proteins, GST-Ehrac1 and GST-Ehrac1-c (catalytic domain), were purified and found to exhibit similar protein kinase activities. The Ehrac1 fusion kinase was found to phosphorylate serine/threonine residues exclusively in vitro. The preferred substrate for the enzyme was histone H1 with a Km of approx. 14 microM. Histone H3 and kemptide were phosphorylated at about half the rate of histone H1. Protamine, enolase, bovine serum albumin, and poly (Glu:Tyr) were not substrates for the enzyme. The protein kinase activity was higher in the presence of Mn2+ than Mg2+. Neither cAMP, Ca2+, nor Ca2+/calmodulin stimulated enzyme activity. The pH optimum of the enzyme was 7.5. The Ehrac1 kinase can utilize GTP as well as ATP as a phosphate donor with an apparent Km of 80 microM. Enzyme activity was inhibited 30-40% by a crude cAMP-dependent protein kinase inhibitor from rabbit and by thiol reagents. The expression and purification of enzymatically active Ehrac1 protein kinase should allow further analysis of the regulation and signal transduction pathways of E. histolytica.
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PMID:Expression and characterization of a rac family protein kinase of Entamoeba histolytica. 798 73

Previously, we have reported the molecular cloning of ara genes encoding a small GTP-binding protein from Arabidopsis thaliana. The criterion based on amino acid sequences suggest that such an ara gene family can be classified to be of the YPT/rab type. To examine the biochemical properties of ARA proteins, several deletions and point mutations were introduced into ara cDNAs. Mutant proteins were expressed in E. coli as GST-chimeric molecules and analyzed in terms of their GTP-binding or GTP-hydrolysing ability in vitro. The results indicate that four conserved amino acid sequence regions of ARA proteins are necessary for GTP-binding. A point mutation of Asn at position 72 for ARA-2, or 71 for ARA-4, to Ile decreased GTP-binding and a point mutation of Gln at position 126 for ARA-2, or 125 for ARA-4, to Leu suppressed GTP-hydrolysis activity. Furthermore, certain factors associated with the membrane fraction accelerated GTPase activities of ARA proteins, suggesting the presence of GTPase activating protein(s) (GAP(s)) in the vesicular transport system of higher plant cells.
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PMID:In vitro mutation analysis of Arabidopsis thaliana small GTP-binding proteins and detection of GAP-like activities in plant cells. 801 29

The Ras-like GTPase Cdc42 is essential for cell polarity and bud site assembly in Saccharomyces cerevisiae by regulating cell cycle-dependent reorganization of cortical cytoskeletal elements. However, its role in mammalian cells is unknown. To identify potential effectors of Cdc42Hs, we incubated lysates from NIH 3T3 fibroblasts or PC12 cells with immobilized glutathione S-transferase (GST)-Cdc42Hs fusion proteins bound to different guanine nucleotides and observed a specific association between the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase) and GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-bound GST-Cdc42Hs. Recombinant p85 formed a complex with GTP gamma S-bound GST-Cdc42Hs and with a GTPase-defective GTP-bound GST-Cdc42Hs-Q61L mutant, but not with a GTP gamma S-bound, effector domain GST-Cdc42HsT35A mutant. Both the Rho-GAP homology domain of p85 and the Cdc42Hs-GAP competitively inhibited the binding of recombinant p85 to Cdc42Hs. In addition, PI 3-kinase activity immunoprecipitated from cell lysates with anti-p85 antibody was stimulated 2-4-fold by GST-Cdc42-GTP gamma S. Similar interactions were observed between p85 and GST-Rac1-GTP gamma S but not between p85 and GST-RhoA-GTP gamma S. These findings suggest that PI 3-kinase, through the Rho-GAP homology domain of p85, can couple to the effector domain of Cdc42Hs and that p85 may be a target for the GTP-bound forms of Cdc42Hs and Rac1.
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PMID:Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. 803 24

Specific binding sites for [3H]leukotriene (LT)D4 and [3H]LTC4 have been identified in sheep lung parenchymal membranes. [3H] LTD4 specific binding was of high affinity (KD = 0.56 nM), saturable (Bmax = 43 fmol/mg of protein), stimulated by divalent cations and inhibited by nonhydrolyzable GTP analogs. LTs and LTD4-receptor antagonists competed for [3H]LTD4 specific binding with the rank order of potency predicted for the LTD4 receptor: LTD4 > ONO-1078 > ICI 204,219 > MK-571 > LTE4 > LTC4 > BAY u9773 >> LTB4. In contrast, [3H]LTC4 specific binding was of lower affinity (KD = 27 nM), abundant (Bmax = 87 pmol/mg of protein) and although stimulated by divalent cations was unaffected by GTP analogs. LTs and LTC4 analogs competed for [3H]LTC4 specific binding with the following rank order of potency: LTC2 > LTC3 > LTC4 > LTC5 >> N-methyl-LTC4 >> LTD4 approximately LTB4 approximately LTB4. [3H]LTD4 specific binding to sheep lung membranes has, therefore, the characteristics of being to a G-protein-coupled LTD4 receptor, whereas the profile of [3H]LTC4 specific binding strongly suggests that these sites are not LT-receptor related. Photolabeling of sheep lung membranes using [125I]azido-LTC4, a photoactivable LTC4 analog, resulted in the selective photolabeling of two polypeptides migrating at 30 kDa and 19 kDa. The selective photolabeling of the 19 kDa polypeptide could be modulated in an identical manner to [3H]LTC4 specific binding. This protein is, therefore, a candidate for being the principal [3H]LTC4 specific site in sheep lung membranes and has a comparable molecular mass to microsomal glutathione S-transferase, recently shown to be the predominant LTC4 binding protein in cellular membranes.
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PMID:Characterization of specific binding sites for cysteinyl leukotrienes in sheep lung. 803 38

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

Clostridium difficile toxin B exhibits cytotoxic activity that is characterized by the disruption of the microfilamental cytoskeleton. Here we studied whether the GTP-binding Rho protein, which reportedly participates in the regulation of the actin cytoskeleton, is involved in the toxin action. Toxin B treatment of Chinese hamster ovary cells reveals a time- and concentration-dependent decrease in the ADP-ribosylation of Rho by Clostridium botulinum C3 exoenzyme in the cell lysate. Disruption of the microfilament system induced by C. botulinum C2 toxin or cytochalasin D does not cause impaired ADP-ribosylation of Rho. Toxin B exhibits its effects on Rho not only in intact cells but also when added to cell lysates. Besides endogenous Rho, RhoA-glutathione S-transferase (Rho-GST) fusion protein added to cell lysate showed decreased ADP-ribosylation after toxin B treatment. Immunoblot analysis reveals identical amounts of Rho-GST and no change in molecular mass after toxin B treatment compared with controls. ADP-ribosylation of Rho-GST purified from toxin B-treated cell lysate is inhibited, indicating a modification of Rho itself. Finally, transfection of rhoA DNA under the control of a strong promoter into cells protects them from the activity of toxin B. Altogether, the data indicate that C. difficile toxin B acts directly or indirectly on Rho proteins to inhibit ADP-ribosylation and suggest that the cytotoxic effect of toxin B involves Rho.
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PMID:Clostridium difficile toxin B acts on the GTP-binding protein Rho. 814 60

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

The Bem2 and Bem3 proteins, which appear to play roles in the regulation of bud site formation in Saccharomyces cerevisiae, show striking homology to a number of proteins that compose a family of GTPase-activating proteins (GAPs) for the rho-subgroup of ras-related GTP-binding proteins. These members include human platelet GAP for Cdc42Hs (the human homolog of a S. cerevisiae GTP-binding protein that regulates bud site assembly), the break point cluster region protein, the brain protein chimerin, the 85-kDa regulatory subunit (p85) of the phosphatidylinositol 3-kinase, and the ras-GAP-binding protein (p190). A fusion protein composed of the glutathione S-transferase protein and the rho-GAP homology region of Bem3 (designated GST-Bem3) stimulates the GTPase activity of the wild-type Cdc42Hs protein (Cdc42HsGly-12), but has no stimulatory effect on a GTPase-defective mutant (Cdc42HsVal-12), whereas a GST-Bem2 fusion protein does not stimulate the GTPase activity of either form of Cdc42Hs. We have compared the ability of GST-Bem3 to serve as a GAP for Cdc42Hs relative to other members of the rho-GAP subfamily and found the following order of potency: human platelet Cdc42Hs GAP > p190 > Bem3 > break point cluster region protein, whereas p85, like Bem2, shows no GAP activity or any ability to bind to the GTP-bound form of Cdc42Hs. We have taken advantage of the functional specificity exhibited by Bem3 (versus Bem2) in using Bem2/Bem3 chimeras, as well as different deletion mutant versions of the Bem3 protein, to delineate the limits of a functional Cdc42 GAP domain. The results of this study indicate that the carboxyl-terminal approximately 224 amino acids (which contain three regions of homology to the other members of the rho-GAP family) represent a "limit GAP." The first two appear to be important for binding to Cdc42Hs and for partial GAP activity.
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PMID:Biochemical comparisons of the Saccharomyces cerevisiae Bem2 and Bem3 proteins. Delineation of a limit Cdc42 GTPase-activating protein domain. 822 21

It has recently been shown that Ras proteins interact directly with Raf serine/threonine kinases in vitro and in the yeast two-hybrid system, leading to speculation that Raf proteins function as effectors for Ras. Here it is demonstrated that the endogenous Raf-1 protein co-immunoprecipitates with Ras from mammalian cells when the non-neutralizing anti-Ras monoclonal antibody Y13-238 is used. The formation of a Ras-Raf complex is absolutely dependent on prior treatment of the cells with a stimulus that activates Ras: phorbol ester or anti-T cell receptor antibody in the case of human peripheral blood T lymphoblasts, or epidermal growth factor in the case of Rat-1 fibroblasts. Up to 3% of cellular Raf-1 can be found in association with Ras. The association is not competed by addition of exogenous GST-Raf to the cell lysates and is therefore unlikely to be due to Ras-Raf binding after cell lysis. Specific interaction of Ras and Raf therefore occurs in intact mammalian cells in response to stimuli that cause Ras to become GTP-bound.
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PMID:Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation. 830 46


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