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)

A disruption of calcium homeostasis, leading to a sustained increase in cytosolic calcium levels, has been associated with cytotoxicity in response to a variety of agents in different cell types. We have observed that administration of a single high dose or multiple lower doses of the carcinogenic nephrotoxin ochratoxin A (OTA) to rats resulted in an increase of the renal cortex endoplasmic reticulum ATP-dependent calcium pump activity. The increase was very rapid, being evident within 10 min of OTA administration and remained elevated for at least 6 hr thereafter. The increase in calcium pump activity was inconsistent with previous observations that OTA enhances lipid peroxidation (ethane exhalation) in vivo, a condition known to inhibit the calcium pump. However, no evidence of enhanced lipid peroxidation was observed in the renal cortex since levels of malondialdehyde and a variety of antioxidant enzymes including catalase, DT-diaphorase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione S-transferase were either unaltered or reduced. In in vitro studies, addition of OTA to cortex microsomes during calcium uptake inhibited the uptake process although the effect was reversible. Preincubation of microsomes with NADPH had a profound inhibitory effect on calcium uptake but inclusion of OTA was able to reverse the inhibition. Changes in the rates of microsomal calcium uptake correlated with changes in the steady-state levels of the phosphorylated Mg2+/Ca(2+)-ATPase intermediate, suggesting that in vivo/in vitro conditions were affecting the rate of enzyme phosphorylation.
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PMID:Alterations in ATP-dependent calcium uptake by rat renal cortex microsomes following ochratoxin A administration in vivo or addition in vitro. 141 61

The ability of the plant phenol ellagic acid to inhibit the mutagenicity of the food mutagen IQ was evaluated using Salmonella typhimurium strain TA98 in the Ames mutagenicity test. Ellagic acid caused a concentration-dependent decrease in the S-9- and microsome-mediated mutagenicity of IQ. The plant phenol did not interact directly with the IQ-derived mutagenic species and did not modify the cytosol-mediated activation of the promutagen. At the concentrations used in the mutagenicity studies, ellagic acid failed to inhibit microsomal mixed-function oxidase activity, including that mediated by the P450I family responsible for the bioactivation of IQ, despite being an essentially planar molecule as indicated by computer-graphic analysis. The inhibitory effect of ellagic acid was independent of its ability to chelate Mg2+. However, pre-incubation of ellagic acid with the bacteria, followed by removal of the plant phenol, did not completely prevent the inhibitory effect of the phenol on the mutagenicity of IQ. Intraperitoneal administration of ellagic acid to rats caused a decrease in total cytochrome P-450 levels and related activities as well as in cytosolic glutathione S-transferase activity. Finally, the possibility that the reported anticarcinogenic action of ellagic acid reflects nothing more than non-selective destruction of hepatic cytochromes P-450, and thus reduced chemical activation, is considered.
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PMID:Antimutagenicity of ellagic acid towards the food mutagen IQ: investigation into possible mechanisms of action. 162 64

Osteopontin (OPN) is an extracellular matrix protein that supports osteoclast adhesion to the bone by binding to integrin alpha v beta 3. We measured the binding between OPN and integrin alpha v beta 3 with recombinant human OPN and the urinary form of human OPN, uropontin. Recombinant OPN was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and cleaved from glutathione S-transferase with Factor Xa. The mass of this form of OPN (rOP27) is 27,046 Da. rOP27 is truncated at arginine residue 228, 69 amino acids short of the native carboxyl terminus. Uropontin and rOP27 support RGD-dependent cell adhesion and to bind purified integrin alpha v beta 3 with similar affinities. Further study showed that OPN is the only known naturally occurring RGD-containing protein with a much greater affinity for alpha v beta 3 than for the platelet integrin alpha IIb beta 3. Most importantly, we find that physiologic levels of Ca2+ block cell adhesion to OPN. Measurement of binding constants between rOPN and purified integrin alpha v beta 3 with surface plasmon resonance showed that the affinity between rOPN and alpha v beta 3 is 26-fold lower in Ca2+ (Kd = 1.1 x 10(-8) M) than in Mn2+ (Kd = 4.3 x 10(-10) M) and 9-fold lower than in Mg2+ (Kd = 1.3 x 10(-9) M). In bone, the resorbing osteoclast generates elevated levels of extracellular Ca2+, therefore the findings presented here suggest a previously unappreciated mechanism for the modulation of bone resorption by extracellular Ca2+.
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PMID:Ca2+ suppresses cell adhesion to osteopontin by attenuating binding affinity for integrin alpha v beta 3. 753 71

Osteopontin (OPN) is an extracellular matrix protein that binds to integrin alpha v beta 3. Here we demonstrate that two other integrins, alpha v beta 1 and alpha v beta 5, are also receptors for OPN. Human embryonic kidney 293 cells adhere to human recombinant osteopontin (glutathione S-transferase-osteopontin; GST-OPN) using integrin alpha v beta 1. When the 293 cells are transfected with the beta 5 subunit, they can also adhere to GST-OPN using integrin alpha v beta 5. Divalent cations regulate the binding of GST-OPN to both alpha v beta 1 and alpha v beta 5. Mg2+ and Mn2+ support the binding of GST-OPN to these integrins but Ca2+ does not. The highest affinity is observed in Mn2+. In the presence of this ion, the affinity of GST-OPN for alpha v beta 1 is 18 nM and the affinity for alpha v beta 5 is 48 nM. The antibody 8A2, which is an agonist for beta 1, promotes the adhesion of 293 cells to GST-OPN even when Ca2+ is present. This observation suggests that cellular events could modulate the affinity of alpha v beta 1 for OPN. Collectively, these findings prove that integrins alpha v beta 1, alpha v beta 3, and alpha v beta 5 have similar affinity for OPN. Therefore, all three integrins must be considered when evaluating the biological affects of OPN.
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PMID:A biochemical characterization of the binding of osteopontin to integrins alpha v beta 1 and alpha v beta 5. 759 29

The alpha subunits of the leukocyte CD11/CD18 integrins contain an approximately 200 amino acid 'inserted' or I domain. The I domain of the cell-surface Mac-1 (CD11b/CD18) integrin has been shown to be the major recognition site for several adhesion ligands, including iC3b, fibrinogen, factor X, and ICAM-1. The I domain from the Mac-1 alpha subunit has been expressed in Escherichia coli as a soluble GST-fusion protein containing a factor Xa sensitive cleavage site. Analytical characterization of the purified I domain reveals that it is obtained in very high quality at high yields. CD and NMR spectra indicate that I domain adopts a predominantly folded structure in solution, independent of the remainder of the alpha subunit. Addition of Ca2+ and Mg2+ did not significantly perturb the structural conformation.
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PMID:Purification and structural characterization of the CD11b/CD18 integrin alpha subunit I domain reveals a folded conformation in solution. 764 57

A novel affinity chromatography purification for human leukotriene C4 synthase is described. It is based on a specific interaction between leukotriene C4 synthase and microsomal glutathione S-transferase which occurs in the presence of magnesium ion. Microsomal glutathione S-transferase was immobilized on NHS-activated Sepharose 4B and used as an affinity matrix. Microsomes from 12-O-tetradecanoyl phorbol 13-acetate-treated human erythroleukemia cells were solubilized with taurocholic acid and applied on the affinity matrix at 0.1 M Mg2+ concentration. After washing with a buffer containing Mg2+, the enzyme was eluted with a glutathione-containing buffer lacking Mg2+. This facile one-step procedure gave a 166-fold purification of leukotriene C4 synthase with a yield of 44%. Analyses of proteins specifically adsorbed to the affinity matrix revealed components with apparent molecular weights of 18, 37, 48, and 60 kDa.
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PMID:Protein-protein interaction affinity chromatography of leukotriene C4 synthase. 766 72

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

The Shope fibroma virus (SFV) DNA ligase gene has been cloned and sequenced, and the biochemical requirements of the gene product have been determined in vitro. The SFV ligase gene maps to the BamHI L1/L2 boundary and spans 1.7 kb. The gene is predicted to encode a 559-amino-acid protein of M(r) = 63,139 which shares 45% amino acid identity with Orthopoxvirus ligases. The C-terminal two-thirds of the protein appears to encode the catalytic domain and shares distant homology with many ligases. The N-terminal homology is shared between only Orthopoxviruses and Leporipoxviruses and suggests that DNA ligases may be composite structures consisting of two independently evolved protein domains. Although the the gene encodes features characteristic of both early and late poxviral genes, Northern analysis showed that SFV ligase is expressed as a late gene product. In order to prove the identity of the protein it was expressed as a glutathione S-transferase fusion in Escherichia coli, affinity purified, and shown to be a Mg2+.ATP-dependent ligase in vitro. The recombinant protein can also form a covalent ligase.AMP complex characteristic of ATP-dependent DNA ligases. The SFV ligase gene can be disrupted and is thus not essential for viral growth in culture. This was shown by recombining a PCR product, encoding a P7.5 promoter and E. coli guanine phosphoribosyltransferase gene (gpt) into the open reading frame, and selecting for gpt+ viruses. This work provides insights into the evolution of Orthopoxviruses and Leporipoxviruses and strains suitable for a detailed analysis of the role DNA ligases play in poxviral recombination.
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PMID:Characterization of the Shope fibroma virus DNA ligase gene. 803 Feb 29

Tumor necrosis factor (TNF) binds two distinct cell surface receptors designated p60 and p80. Our previous studies indicate that a protein kinase from U-937 cells binds to and phosphorylates the p60 receptor. While the p80 receptor is phosphorylated in vivo, no association of a protein kinase has been described. We employed a fusion protein comprising of glutathione S-transferase and the cytoplasmic domain of the p80 receptor (GST-p80CD) to identify cellular proteins that might associate with this receptor. From 35S- and 32P-labeled cells, a protein of 59 kDa bound specifically to GST-p80CD. In vitro kinase reactions indicated that serine/threonine protein kinase activity associated with GST-p80CD and causes its phosphorylation. Additionally, a 59-kDa phosphoprotein was also identified after kinase reactions of proteins bound to GST-p80CD. This kinase activity required either Mg2+ or Mn2+ for optimal activity, and it phosphorylated myelin basic protein, histone H2B, and also the cytoplasmic domain of the p60 receptor. Treatment of cells with TNF increased the p80 receptor-associated kinase activity by 200%. In summary, our results provide evidence of a novel ligand-activated serine/threonine protein kinase that associates with the cytoplasmic domain of the p80 receptor and causes the phosphorylation of both forms of the TNF receptor. This p80 TNF receptor-associated protein and the associated kinase described here are referred to as p80-TRAP and p80-TRAK, respectively.
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PMID:Physical and functional association of a serine-threonine protein kinase to the cytoplasmic domain of the p80 form of the human tumor necrosis factor receptor in human histiocytic lymphoma U-937 cells. 805 Oct 45

The mouse mammary tumor virus gag-pro transframe protein (p30) contains the nucleocapsid protein domain derived from the 3' end of gag, fused to 154 residues encoded by the 5' region of the pro open reading frame. The DNA coding for p30 was cloned into the plasmid pALTER-1, and an additional nucleotide was inserted by site-directed mutagenesis to allow the read-through from the gag into the pro open reading frame. The obtained insert was then cloned into pGEX-2T, a plasmid containing the glutathione S-transferase gene of Schistosoma japonicum and a nucleotide sequence encoding for a thrombin cleavage site. The chimeric protein (GST-p30) was isolated by affinity chromatography on a glutathione-Sepharose 4B column, and after thrombin treatment, the excised p30 was further purified on a single-stranded DNA-agarose column. This protein showed dUTPase activity, with only negligible cleavage of dATP, dGTP, dCTP, dTTP, or UTP. Its apparent Km for dUTP was 28 microM. The enzyme was inhibited by EDTA, but its effect could be reversed by Mg2+ and other divalent cations. dUTPase activity was also detected in purified mouse mammary tumor virus, and p30 was the only protein recognized by antibodies directed towards the carboxyl-terminal sequence of the dUTPase coding region.
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PMID:Expression and purification of the mouse mammary tumor virus gag-pro transframe protein p30 and characterization of its dUTPase activity. 813 16


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