Identification and characterization of the zebrafish glutathione S‐transferase Pi‐1

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S‐transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi‐1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3‐month‐old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione‐conjugating activity toward 1‐chloro‐2,4‐dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH‐dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.     

Molecular characterization and expression of metallothionein from freshwater pearl mussel,Hyriopsis schlegelii

Two metallothionein genes (HsMT1 and HsMT2) were first identified and described from Hyriopsis schlegelii. The open reading frame of HsMT1 and HsMT2 were 216 and 222 bp, encoding a protein of 71 and 73 amino acid residues. The deduced amino acid sequences showed they contained parts of typical MT characteristics, apart from HsMT2 lacked Cys–Cys motifs. The phylogenetic tree showed HsMT1 shared a high similarity with that of other molluscs, but HsMT2 was split into a distinct group separated from known molluscan MTs. HsMT1 exhibited constitutive expression in all examined tissues and the highest expression occurred in hepatopancreas, however, nearly all HsMT2 was just detected in gonad. After Cd exposure, their mRNA levels presented similar expression patterns. The transgenic bacteria of HsMT1 showed higher tolerance than HsMT2 in Cd environment. It was implied that HsMT1 and HsMT2 were involved in metal response but HsMT2 might have other physiological functions.     

Evaluation of chalcones as inhibitors of glutathione S‐transferase

Glutathione S‐transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play an important role in cellular signaling. In the present study, potential inhibition effects of chalcones were tested against human GST. For this purpose, GST was purified from human erythrocytes with 5.381 EU?mg^?1 specific activity and 51.95% yield using a GSH–agarose affinity chromatographic method. The effects of chalcones on in vitro GST activity were tested at various concentrations. K_i constants of chalcones were found in the range of 7.76–41.93 μM. According to the results, 4‐fluorochalcone showed a better inhibitory effect compared with the other compounds. The inhibition mechanisms of 2'‐hydroxy‐4‐methoxychalcone and 4‐methoxychalcone were noncompetitive, whereas the inhibition mechanisms of 4'‐ hydroxychalcone, 4‐ fluorochalcone, and 4,4'‐ diflurochalcone were competitive.     

Some metals inhibit the glutathione S‐transferase from Van Lake fish gills

Glutathione S‐transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play important role cellular signaling. The present article focuses on the role of Cd²⁺, Cu²⁺, Zn²⁺, and Ag⁺ in vitro inhibition of GST. For this purpose, GST was purified from Van Lake fish (Chalcalburnus tarichii Pallas) gills with 110.664 EU mg^?1 specific activity and 79.6% yield using GSH‐agarose affinity chromatographic method. The metal ions were tested at various concentrations on in vitro GST activity. IC₅₀ values were found for Cd⁺², Cu⁺², Zn⁺², Ag⁺ as 450.32, 320.25, 1510.13, and 16.43 μM, respectively. K _i constants were calculated as 197.05 ± 105.23, 333.10 ± 152.76, 1670.21 ± 665.43, and 0.433 ± 0.251 μM, respectively. Ag⁺ showed better inhibitory effect compared with the other metal ions. The inhibition mechanisms of Cd²⁺ and Cu²⁺ were non‐competitive, whereas Zn²⁺ and Ag⁺ were competitive. Co²⁺, Cr²⁺, Pb²⁺, and Fe³⁺ had no inhibitory activity on GST.     

Characterization of glutathione S‐transferase in the saturniid moth,Samia Cynthia pryeri (Lep.: Saturniidae)

An enzyme, which possesses glutathione S‐transferase (GST) activity, has been found in the midgut of the saturniid moth, Samia cynthia pryeri. The enzyme was initially purified into homogeneity by ammonium sulphate fractionation, affinity chromatography, and ion‐exchange chromatography. The resulting enzyme revealed a single band with a molecular mass of 23 kDa by sodium dodecyl sulfate polyacrylamide electrophoresis under reduced conditions. When tested with 1‐chloro‐2,4‐dinitrobenzene, a universal substrate of GST, the purified remnants had an optimum pH of 8.0 for enzymatic activity, and was fairly stable at pH 5–9 and at temperatures below 40°C. The enzyme was also responsive to 4‐hydroxynonenal, a cytotoxic lipid‐peroxidation product. The present GST was inhibited by organophosphorus and pyrethroid insecticides including fenitrothion, permethrin and deltamethrin.     

Benzyl isothiocyanate attenuates the hydrogen peroxide‐induced interleukin‐13 expression through glutathione S‐transferase P induction in T lymphocytic leukemia cells

We investigated the effect of benzyl isothiocyanate (BITC) on the hydrogen peroxide‐induced gene expression of a T‐helper‐2 cytokine, interleukin (IL)‐13, in T lymphocytic leukemia Jurkat cells. The 24‐h pretreatment of BITC significantly inhibited the IL‐13 expression enhanced by hydrogen peroxide. Although the BITC pretreatment did not change the enhanced level of the phosphorylated c‐Jun N‐terminal kinase (JNK), it significantly inhibited the nuclear translocation of c‐Jun induced by hydrogen peroxide. BITC also increased the protein expression of glutathione S‐transferase (GST) isozymes, GSTP1/2, as well as the total GST activity. A GSTP1/2‐specific inhibitor, 6‐(7‐nitro‐2,1,3‐benzoxadiazol‐4‐ylthio)hexanol (NBDHEX), significantly counteracted the inhibitory effect of BITC on the hydrogen peroxide‐enhanced IL‐13 upregulation as well as the c‐Jun nuclear translocation. Taken together, these results suggested that BITC inhibits the oxidative stress‐mediated IL‐13 mRNA expression, possibly through interference of the c‐Jun phosphorylation by GSTP.     

Detoxification of insecticides,allechemicals and heavy metals by glutathione S‐transferase SlGSTE1 in the gut of Spodoptera litura

Insect glutathione S‐transferases (GSTs) play important roles in detoxifying toxic compounds and eliminating oxidative stress caused by these compounds. In this study, detoxification activity of the epsilon GST SlGSTE1 in Spodoptera litura was analyzed for several insecticides and heavy metals. SlGSTE1 was significantly up‐regulated by chlorpyrifos and xanthotoxin in the midgut of S. litura. The recombinant SlGSTE1 had V_max (reaction rate of the enzyme saturated with the substrate) and K_m (michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme) values of 27.95 ± 0.88 μmol/min/mg and 0.87 ± 0.028 mmol/L for glutathione, respectively, and V_max and K_m values of 22.96 ± 0.78 μmol/min/mg and 0.83 ± 0.106 mmol/L for 1‐chloro‐2,4‐dinitrobenzene, respectively. In vitro enzyme indirect activity assay showed that the recombinant SlGSTE1 possessed high binding activities to the insecticides chlorpyrifos, deltamethrin, malathion, phoxim and dichloro‐diphenyl‐trichloroethane (DDT). SlGSTE1 showed higher binding activity to toxic heavy metals cadmium, chromium and lead than copper and zinc that are required for insect normal growth. Western blot analysis showed that SlGSTE1 was induced in the gut of larvae fed with chlorpyrifos or cadmium. SlGSTE1 also showed high peroxidase activity. All the results together indicate that SlGSTE1 may play an important role in the gut of S. litura to protect the insect from the toxic effects of these compounds and heavy metals.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Purification of fat body glutathione S‐transferase from the desert locust Schistocerca gregaria: investigation of flavonoid inhibitory effects on enzyme activity

Focus on the development of botanical insecticides such as polyphenols may represent an alternative method to chemical control. In the present study, total glutathione concentration and its related antioxidant enzymes in foregut, midgut, hindgut and fat body homogenates of the desert locust Schistocerca gregaria are examined. Glutathione S‐transferase (GST) activity exhibits a significantly higher value in fat bodies compared with other tissues. A simple and reproducible procedure for the purification of S. gregaria fat body GST is established and the purified enzyme is shown to be homogenous. The purified GST displays a typical Michaelis behaviour with respect to its substrates. Characterization of the GST, including optimum pH, substrate specificity and inhibitor effects, is carried out. The ability of some flavonoids to inhibit S. gregaria fat body GST activity is examined. High‐performance liquid chromatography analysis indicates that the major components in Glycyrrhiza glabra roots are 18α‐glycyrrhetinic acid, quercetin and rutin, and the major components in Hibiscus sabdariffa calyx are cyanidin 3‐O‐glucoside chloride and delphinidin. Quercetin and delphinidin chloride exhibit strong GST inhibition and the inhibition type is determined for both. Rutin shows a smaller inhibitory effect, whereas 18α‐glycyrrhetinic acid and cyanidin have no effect. Inhibition of S. gregaria fat body GST activity would be expected to prevent, or at least delay, the development of resistance to chemical pesticides. Among the examined levels of the antioxidant enzymes, total glutathione concentration and its related enzymes in foregut, midgut, hindgut and fat body crude homogenates of S. gregaria GST activity exhibit a significantly higher value in fat bodies compared with other tissues. Some flavonoids that are detected in H. sabdariffa calyx and G. glabra root extracts are the most effective inhibitors of the purified S. gregaria fat body GST activity. Inhibition of S. gregaria fat body GST activity by quercetin and delphinidin (major compounds detected by HPLC) would be expected to prevent, or at least delay, the development of resistance to chemical pesticides.     

The structure of a shellfish specific GST class glutathione S‐transferase from antarctic bivalve Laternula elliptica reveals novel active site architecture

Glutathione‐S‐transferases have been identified in all the living species examined so far, yet little is known about their function in marine organisms. In a previous report, the recently identified GST from Antarctic bivalve Laternula elliptica (LeGST) was classified into the rho class GST, but there are several unique features of LeGST that may justify reclassification, which could represent specific shellfish GSTs. Here, we determined the crystal structure of LeGST, which is a shellfish specific class of GST. The structural analysis showed that the relatively open and wide hydrophobic H‐site of the LeGST allows this GST to accommodate various substrates. These results suggest that the H‐site of LeGST may be the result of adaptation to their environments as sedentary organisms. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Inhibition effects of pesticides on glutathione‐S‐transferase enzyme activity of Van Lake fish liver

Glutathione‐S‐transferases (GSTs) have a function in xenobiotic metabolism. They are a significant multifunctional family with a wide variety of catalytic activities. In the current study, we determined in vitro inhibition effects of 2,4‐dichlorophenoxyacetic acid dimethylamine salt (2,4‐D DMA), haloxyfop‐P‐methyl, glyphosate isopropylamine, dichlorvos, and λ‐cyhalothrin on purified GST. For this purpose, GST were purified from Van Lake fish (Chalcalburnus tarichii Pallas) liver with 29.25 EU mg^?1 specific activity and 10.76% yield using GSH–agarose affinity chromatographic method. The pesticides were tested at various concentrations on in vitro GST activity. K_i constants were calculated as 0.17 ± 0.01, 0.25 ± 0.05, 3.72 ± 0.32, 0.42 ± 0.06, and 0.025 ± 0.004 mM, for 2,4‐D DMA, haloxyfop‐P‐methyl, glyphosate isopropylamine, dichlorvos, and λ‐cyhalothrin, respectively. λ‐Cyhalothrin showed a better inhibitory effect compared to the other pesticides. The inhibition mechanisms of λ‐cyhalothrin were competitive, while the other pesticides were noncompetitive.     

Structures of yeast glutathione‐S‐transferase Gtt2 reveal a new catalytic type of GST family

Glutathione‐S‐transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand‐bound forms, at 2.23 Å, 2.20 Å and 2.10 Å, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues—tyrosine, serine and cysteine—distinguishes it from all other cytosolic GSTs of known structure. Site‐directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino‐terminus of helix‐α1 because of stereo‐hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.