The conjugation of 4-nitroquinoline 1-oxide, a potent carcinogen, by mammalian glutathione transferases. 4-Nitroquinoline 1-oxide conjugation by human, rat and mouse liver cytosols, extrahepatic organs of mice and purified mouse glutathione transferase isoenzymes.
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ABSTRACT:
SUBMITTER: Stanley JS
PROVIDER: S-EPMC1135404 | biostudies-other | 1988 Nov
REPOSITORIES: biostudies-other
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Project description:Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively.
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Project description:Benzene is a known human carcinogen which must be activated to benzene oxide (BO) to exert its carcinogenic potential. BO can be detoxified in vivo by reaction with glutathione and excretion in the urine as S-phenylmercapturic acid. This process may be catalyzed by glutathione S-transferases (GSTs), but kinetic data for this reaction have not been published. Therefore, we incubated GSTA1, GSTT1, GSTM1, and GSTP1 with glutathione and BO and quantified the formation of S-phenylglutathione. Kinetic parameters were determined for GSTT1 and GSTP1. At 37 °C, the putative Km and Vmax values for GSTT1 were 420 μM and 450 fmol/s, respectively, while those for GSTP1 were 3600 μM and 3100 fmol/s. GSTA1 and GSTM1 did not exhibit sufficient activity for determination of kinetic parameters. We conclude that GSTT1 is a critical enzyme in the detoxification of BO and that GSTP1 may also play an important role, while GSTA1 and GSTM1 seem to be less important.
| S-EPMC4695229 | biostudies-literature
Project description:The 55 Arabidopsis glutathione transferases (GSTs) are, with one microsomal exception, a monophyletic group of soluble enzymes that can be divided into phi, tau, theta, zeta, lambda, dehydroascorbate reductase (DHAR) and TCHQD classes. The populous phi and tau classes are often highly stress inducible and regularly crop up in proteomic and transcriptomic studies. Despite much study on their xenobiotic-detoxifying activities their natural roles are unclear, although roles in defence-related secondary metabolism are likely. The smaller DHAR and lambda classes are likely glutathione-dependent reductases, the zeta class functions in tyrosine catabolism and the theta class has a putative role in detoxifying oxidised lipids. This review describes the evidence for the functional roles of GSTs and the potential for these enzymes to perform diverse functions that in many cases are not "glutathione transferase" activities. As well as biochemical data, expression data from proteomic and transcriptomic studies are included, along with subcellular localisation experiments and the results of functional genomic studies.
| S-EPMC3244946 | biostudies-literature
Project description:SummaryThe soluble glutathione transferases (GSTs, EC 2.5.1.18) are encoded by a large and diverse gene family in plants, which can be divided on the basis of sequence identity into the phi, tau, theta, zeta and lambda classes. The theta and zeta GSTs have counterparts in animals but the other classes are plant-specific and form the focus of this article. The genome of Arabidopsis thaliana contains 48 GST genes, with the tau and phi classes being the most numerous. The GST proteins have evolved by gene duplication to perform a range of functional roles using the tripeptide glutathione (GSH) as a cosubstrate or coenzyme. GSTs are predominantly expressed in the cytosol, where their GSH-dependent catalytic functions include the conjugation and resulting detoxification of herbicides, the reduction of organic hydroperoxides formed during oxidative stress and the isomerization of maleylacetoacetate to fumarylacetoacetate, a key step in the catabolism of tyrosine. GSTs also have non-catalytic roles, binding flavonoid natural products in the cytosol prior to their deposition in the vacuole. Recent studies have also implicated GSTs as components of ultraviolet-inducible cell signaling pathways and as potential regulators of apoptosis. Although sequence diversification has produced GSTs with multiple functions, the structure of these proteins has been highly conserved. The GSTs thus represent an excellent example of how protein families can diversify to fulfill multiple functions while conserving form and structure.
| S-EPMC139027 | biostudies-literature
Project description:In humans, the glutathione S-transferases (GST) protein family is composed of seven members that present remarkable structural similarity and some degree of overlapping functionalities. GST proteins are crucial antioxidant enzymes that regulate stress-induced signaling pathways. Interestingly, overactive GST proteins are a frequent feature of many human cancers. Recent evidence has revealed that the biology of most GST proteins is complex and multifaceted and that these proteins actively participate in tumorigenic processes such as cell survival, cell proliferation, and drug resistance. Structural and pharmacological studies have identified various GST inhibitors, and these molecules have progressed to clinical trials for the treatment of cancer and other diseases. In this review, we discuss recent findings in GST protein biology and their roles in cancer development, their contribution in chemoresistance, and the development of GST inhibitors for cancer treatment.
| S-EPMC8146591 | biostudies-literature
Project description:Glutathione S-transferases are a complex family of dimeric proteins that play a dual role in cellular detoxification; they catalyse the first step in the synthesis of mercapturic acids, and they bind potentially harmful non-substrate ligands. Bile acids are quantitatively the major group of ligands encountered by the glutathione S-transferases. The enzymes from rat liver comprise Yk (Mr 25 000), Ya (Mr 25 500), Yn (Mr 26 500), Yb1, Yb2 (both Mr 27 000) and Yc (Mr 28 500) monomers. Although bile acids inhibited the catalytic activity of all transferases studied, the concentration of a particular bile acid required to produce 50% inhibition (I50) varies considerably. A comparison of the I50 values obtained with lithocholate (monohydroxylated), chenodeoxycholate (dihydroxylated) and cholate (trihydroxylated) showed that, in contrast with all other transferase monomers, the Ya subunit possesses a relatively hydrophobic bile-acid-binding site. The I50 values obtained with lithocholate and lithocholate 3-sulphate showed that only the Ya subunit is inhibited more effectively by lithocholate than by its sulphate ester. Other subunits (Yk, Yn, Yb1 and Yb2) were inhibited more by lithocholate 3-sulphate than by lithocholate, indicating the existence of a significant ionic interaction, in the bile-acid-binding domain, between (an) amino acid residue(s) and the steroid ring A. By contrast, increasing the assay pH from 6.0 to 7.5 decreased the inhibitory effect of all bile acids studied, suggesting that there is little significant ionic interaction between transferase subunits and the carboxy group of bile acids. Under alkaline conditions, low concentrations (sub-micellar) of nonsulphated bile acids activated Yb1, Yb2 and Yc subunits but not Yk, Ya and Yn subunits. The diverse effects of the various bile acids studied on transferase activity enables these ligands to be used to help establish the quaternary structure of individual enzymes. Since these inhibitors can discriminate between transferases that appear to be immunochemically identical (e.g. transferases F and L), bile acids can provide information about the subunit composition of forms that cannot otherwise be distinguished.
| S-EPMC1153041 | biostudies-other
Project description:In addition to their well-established role in detoxication, glutathione transferases (GSTs) have other biological functions. We are focusing on the ketosteroid isomerase activity, which appears to contribute to steroid hormone biosynthesis in mammalian tissues. A highly efficient GST A3-3 is present in some, but not all, mammals. The alpha class enzyme GST A3-3 in humans and the horse shows the highest catalytic efficiency with kcat/Km values of approximately 107 M-1s-1, ranking close to the most active enzymes known. The expression of GST A3-3 in steroidogenic tissues suggests that the enzyme has evolved to support the activity of 3β-hydroxysteroid dehydrogenase, which catalyzes the formation of 5-androsten-3,17-dione and 5-pregnen-3,20-dione that are substrates for the double-bond isomerization catalyzed by GST A3-3. The dehydrogenase also catalyzes the isomerization, but its kcat of approximately 1 s-1 is 200-fold lower than the kcat values of human and equine GST A3-3. Inhibition of GST A3-3 in progesterone-producing human cells suppress the formation of the hormone. Glutathione serves as a coenzyme contributing a thiolate as a base in the isomerase mechanism, which also involves the active-site Tyr9 and Arg15. These conserved residues are necessary but not sufficient for the ketosteroid isomerase activity. A proper assortment of H-site residues is crucial to efficient catalysis by forming the cavity binding the hydrophobic substrate. It remains to elucidate why some mammals, such as rats and mice, lack GSTs with the prominent ketosteroid isomerase activity found in certain other species. Remarkably, the fruit fly Drosophila melanogaster, expresses a GSTE14 with notable steroid isomerase activity, even though Ser14 has evolved as the active-site residue corresponding to Tyr9 in the mammalian alpha class.
| S-EPMC8645602 | biostudies-literature
Project description:UnlabelledBiliary atresia, the most common indication for pediatric liver transplantation, is a fibrotic disease of unknown etiology affecting the extrahepatic bile ducts of newborns. The recently described toxin biliatresone causes lumen obstruction in mouse cholangiocyte spheroids and represents a new model of biliary atresia. The goal of this study was to determine the cellular changes caused by biliatresone in mammalian cells that ultimately lead to biliary atresia and extrahepatic fibrosis. We treated mouse cholangiocytes in three-dimensional (3D) spheroid culture and neonatal extrahepatic duct explants with biliatresone and compounds that regulate glutathione (GSH). We examined the effects of biliatresone on SOX17 levels and determined the effects of Sox17 knockdown on cholangiocytes in 3D culture. We found that biliatresone caused disruption of cholangiocyte apical polarity and loss of monolayer integrity. Spheroids treated with biliatresone had increased permeability as shown by rhodamine efflux within 5 hours compared with untreated spheroids, which retained rhodamine for longer than 12 hours. Neonatal bile duct explants treated with the toxin showed lumen obstruction with increased subepithelial staining for α-smooth muscle actin and collagen, consistent with fibrosis. Biliatresone caused a rapid and transient decrease in GSH, which was both necessary and sufficient to mediate its effects in cholangiocyte spheroid and bile duct explant systems. It also caused a significant decrease in cholangiocyte levels of SOX17, and Sox17 knockdown in cholangiocyte spheroids mimicked the effects of biliatresone.ConclusionBiliatresone decreases GSH and SOX17 in mouse cholangiocytes. In 3D cell systems, this leads to cholangiocyte monolayer damage and increased permeability; in extrahepatic bile duct explants, it leads to disruption of the extrahepatic biliary tree and subepithelial fibrosis. This mechanism may be important in understanding human biliary atresia. (Hepatology 2016;64:880-893).
| S-EPMC4992464 | biostudies-literature
Project description:The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.
| S-EPMC1148470 | biostudies-other
Project description:The hepatic glutathione S-transferases (Gsts) are critical phase II enzymes in protecting cellular macromolecules against electrophiles and oxidative stress. Little is known about the ontogeny of Gsts and the underlying regulatory mechanisms during liver development. Therefore, in this study, the ontogeny and the regulatory mechanisms of 19 known Gst isoforms were investigated in mouse liver from 2 days before birth to postnatal day 45. With the exception of Gstm5 and MGst2 that showed a progressive decline in postnatal messenger RNA (mRNA) expression, most other Gst isoforms showed a progressive increase in postnatal mRNA expression. Two-way hierarchical clustering revealed three distinct expression patterns of these Gsts isoforms: perinatal, adolescent, and adult enriched. The expression signatures of certain Gst isoforms showed positive association with the ontogeny of critical xenobiotic-sensing transcription factors, including aryl hydrocarbon receptor, pregnane X receptor (PXR), constitutive androstane receptor, peroxisome proliferator-activated receptor alpha, and NF-E2-related factor-2. Specifically, genome-wide chromatin immunoprecipitation coupled with the next generation sequencing technology (ChIP-Seq) revealed direct PXR-binding sites to the Gsta, Gstm, Gstt, and Gstp polycistron clusters as well as to the Mgst1 gene locus. Chromatin immunoprecipitation-on-chip analysis demonstrated that DNA methylation and histone H3K27-trimethylation (H3K27me3), two-gene expression-suppressing epigenetic marks, were consistently low around the Gstz1 gene locus. In contrast, enrichment of histone H3K4-dimethylation (H3K4me2), a hallmark for gene activation, increased 60% around the Gstz1 gene locus from prenatal to the young adult period. Regression analysis revealed a strong correlation between the enrichment of H3K4me2 and Gstz1 mRNA expression (r = 0.76). In conclusion, this study characterized three distinct ontogenic expression signatures of the 19 Gst isoforms and examined some genetic and epigenetic mechanisms inducing their transcription during liver development.
| S-EPMC2886863 | biostudies-literature