• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.647-650
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• 2003

In anhydrous organic solvents, the complexes formed between AOT (dodecylbenzene sulfuric acid sodium salt) and hemoproteins, such as hemoglobin, myoglobin, or cytochrome c, displayed remarkably higher activity than the hemoprotein powders to oxidize dibenzothiophene, a model compound of organic sulfurs contained in fossil fuels. In slightly hydrophobic organic solvents, such as ethyl acetate and butyl acetate, dibenzothiophene was completely oxidized catalytically by the cytochrome c-AOT complex with cumene hydroperoxide (${\alpha},{\alpha}-dimethylbenzyl$ hydroperoxide) as an oxidant. In highly hydrophobic organic solvents, such as decane and hexadecane, however, the activity of the cytochrome c-AOT complex decreased, presumably due to the aggregation of the hemoprotein-AOT complex in these solvents.

• Analytical Science and Technology
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• v.22 no.3
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• pp.228-234
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• 2009

To gain further insight into herbicide detoxification of plant, we purified a glutathione S-transferase from Brassica oleracea (BoGST) and studied its substrate specificity towards several xenobiotic compounds. The BoGST was purified to electrophoretic homogeneity with approximately 10% activity yield by DEAE-Sephacel and GSHSepharose column chromatography. The molecular weight of the BoGST was determined to be approximately 23,000 by SDS-polyacrylamide gel electrophoresis and 48,000 by gel chromatography, indicating a homodimeric structure. The activity of the BoGST was significantly inhibited by S-hexyl-GSH and S-(2,4-dinitrophenyl)GSH. The substrate specificity of the BoGST displayed high activities towards CDNB, a general GST substrate and ethacrynic acid. It also exhibited GSH peroxidase activity toward cumene hydroperoxide.

• Applied Chemistry for Engineering
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• v.3 no.4
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• pp.627-638
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• 1992

PPG and BPA-type polyurethane prepolymers(NCO terminated) were obtained from bisphenol A and four types of PPG, having different molecular weight and numbers of functional groups. PPG and BPA-type polyurethane dimethacrylates were synthesized by reacting PPG and BPA-type polurethane prepolymer with 2-HEMA respectively. PPG-type polyurethane dimethacrylates were formulated with initiator(CHP), inhibitor(hydroquinone) and reactive diluent(TEGDMA). The effect of formulation on the torque changes was studied. Four kinds of PPG-type polyurethane dimethacrylates, having different molecular weight and numbers of functional group, were formulated with the same amount of CHP, TEGDMA and hydroquinone. The effects of the molecular weight of PPG-type polyurethane dimethacrylate and functional group numbers of PPG-type polyurethane dimethacrylates on the torque were investigated. These results showed that the torque of PPG-type polyurethane dimethacrylates, having same numbers of functional group, increased with decreasing molecular weight of dimethacrylates and torque of PPG-type polyurethane dimethacrylate, having similar molecular weight, was increased with increasing the number of fuctional group. The glass-transition temperature(Tg) of gels obtained by thermosetting cure for the four kinds of PPG-type polyurethane dimethacrylates were measured by DSC and molecular weights between cross-links(Mc) were calculated from Tg changes.

• BMB Reports
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• v.38 no.2
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• pp.232-237
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• 2005

A glutathione S-transferase (GST) from Lactuca sativa was purified to electrophoretic homogeneity approximately 403-fold with a 9.6% activity yield by DEAE-Sephacel and glutathione (GSH)-Sepharose column chromatography. The molecular weight of the enzyme was determined to be approximately 23,000 by SDS-polyacrylamide gel electrophoresis and 48,000 by gel chromatography, indicating a homodimeric structure. The activity of the enzyme was significantly inhibited by S-hexylGSH and S-(2,4-dinitrophenyl) glutathione. The enzyme displayed activity towards 1-chloro-2,4-dinitrobenzene, a general GST substrate and high activities towards ethacrynic acid. It also exhibited glutathione peroxidase activity toward cumene hydroperoxide.

• Molecules and Cells
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• v.40 no.4
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• pp.299-306
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• 2017

The transcriptional activator AphB has been implicated in acid resistance and pathogenesis in the food borne pathogens Vibrio vulnificus and Vibrio cholerae. To date, the full-length AphB crystal structure of V. cholerae has been determined and characterized by a tetrameric assembly of AphB consisting of a DNA binding domain and a regulatory domain (RD). Although acidic pH and low oxygen tension might be involved in the activation of AphB, it remains unknown which ligand or stimulus activates AphB at the molecular level. In this study, we determine the crystal structure of the AphB RD from V. vulnificus under aerobic conditions without modification at the conserved cysteine residue of the RD, even in the presence of the oxidizing agent cumene hydroperoxide. A cysteine to serine amino acid residue mutant RD protein further confirmed that the cysteine residue is not involved in sensing oxidative stress in vitro. Interestingly, an unidentified small molecule was observed in the inter-subdomain cavity in the RD when the crystal was incubated with cumene hydroperoxide molecules, suggesting a new ligand-binding site. In addition, we confirmed the role of AphB in acid tolerance by observing an aphB-dependent increase in cadC transcript level when V. vulnificus was exposed to acidic pH. Our study contributes to the understanding of the AphB molecular mechanism in the process of recognizing the host environment.

• BMB Reports
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• v.31 no.4
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• pp.399-404
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• 1998

In order to gain further insight on the relationship between structure and function of glutathione S-transferase (GST), the six active-site mutants, R13T, K44T, Q51A, Q64A, S65A, and D98A, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The active-site mutants showed marked differences in substrate specificity. The substitution of Gln51 with threonine resulted in a drastic decrease in the specific activities to <10% of the wild-type value. The substitution of Arg13 with threonine resulted in more decreased specific activity toward cumene hydroperoxide and in the $I_{50}$ values of S-(2,4-dinitrophenyl) glutathione and benanstatin A. These results suggest that the substitution of Arg13 with threonine changes the conformation of the active site to increase the affinity for the product or electrophilic substrate. Lys44 seems to be in the vicinity of the H-site of hGST P1-1 or may contribute to some extents to the electrophile binding.

• BMB Reports
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• v.40 no.4
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• pp.511-516
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• 2007

A glutathione S-transferase (GST) related to the phi (F) class of enzymes only found in plants has been cloned from the Oryza sativa. The GST cDNA was cloned by PCR using oligonucleotide primers based on the OsGSTF5 (GenBank Accession No. $\underline{AF309382}$) sequences. The cDNA was composed of a 669-bp open reading frame encoding for 223 amino acids. The deduced peptide of this gene shared on overall identity of 75% with other known phi class GST sequences. On the other hands, the OsGSTF5 sequence showed only 34% identity with the sequence of the OsGSTF3 cloned by our previous study (Cho et al., 2005). This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF5 formed a homo-dimer composed of 28 kDa subunit and its pI value was approximately 7.8. The expressed OsGSTF5 displayed glutathione conjugation activity toward 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane and glutathione peroxidase activity toward cumene hydroperoxide. The OsGSTF5 also had high activities towards the herbicides alachlor, atrazine and metolachlor. The OsGSTF5 was highly sensitive to inhibition by S-hexylGSH, benastatin A and hematin. We propose from these results that the expressed OsGSTF5 is a phi class GST and appears to play a role in the conjugation of herbicide and GPOX activity.

• Bulletin of the Korean Chemical Society
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• v.26 no.3
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• pp.433-439
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• 2005

To gain further insight on the relationship between structure and functions of glutathione S-transferase (GST), the three tyrosine 108 mutants, Y108A, Y108F, and Y108W, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitution of Tyr 108 with alanine resulted in significant decrease of the GSH-conjugation activity and the GSH peroxidase activity, but approximately 63% increase of steroid isomerase activity toward ${\Delta}^5$–[androstene 3,17-dione. On the other hand, the substitution of Tyr 108 with phenylalanine resulted in decreases of $k_{cat}\;and\;k_{cat}/K_m{^{EPNP}}$ by 2 orders of magnitude, suggesting that Tyr 108 residue of hGSTP1-1 are considered to be important for the catalysis and the binding of the epoxide substrates. The substitution of Tyr 108 with tryptophan resulted in significant decreases of the specific activities toward EPNP, cumene hydroperoxide and ${\Delta}^5$–ndrostene 3,17-dione, but approximately 2-fold increase on the enzyme-catalyzed addition of GSH to DCNB. We conclude from these results that Tyr 108 in hGST P1-1 plays very different roles depending upon the nature of the electrophilic substrates.

• Journal of Environmental Health Sciences
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• v.30 no.3
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• pp.230-238
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• 2004

The adverse health effects of a number of environment pollutions are related to the formation of free radicals. Induction of antioxidant defensive system in the response to an oxidative attack is an essential element of the cell to survive. CYP2E1 is easily induced by organic solvents and induces continuous formation of reactive oxygen species (ROS). ${\gamma}$-Glutamyltranspeptidase (${\gamma}$GT) plays an important role in glutathione metabolism and xenobiotic detoxification. To evaluate the characteristic of oxidative stress which induces GGT expression and to understand human antioxidant defensive response against oxidative stress induced by CYP2E1, we studied regulation of ${\gamma}$GT enzyme expression in response to various oxidative stresses in human HepG2 cells. The ${\gamma}$GT activity was not modified after exposure of acute oxidative stress inducing agents (ferric nitrilotriacetate, cumene hydroperoxide, ADP-Fe, O-tetradecanoylphorbol-13-acetate, tumor necrosis factor-alpha). To induce continuous exposure of cells to ROS, HepG2 cells were transfected by human CYP2E1 gene transiently. The CYP2E1 activity was verified with chlorzoxazone hydroxylation. Transfection of CYP2E1 showed continuous 60% increase in intracellular ROS and 240 % increase in microsomal ROS. CYP2E1 overexpressing cells showed increased ${\gamma}$GT activity (2.5-fold). The observed enhancement of ${\gamma}$GT activity correlated with a significant increase of ${\gamma}$GT mRNA (2.1-fold). Treatment with antioxidant strongly prevented the increase in ${\gamma}$GT activity. The CYP2E1 overexpression did not modify toxicity index and increased glutathione levels. These results show that continuous exposure of cells to ROS produced by CYP2E1 up-regulates ${\gamma}$GT; This may be one of the adaptive antioxidant responses of cells to oxidative insult. Present study also suggests that the induction of ${\gamma}$GT could be used as a marker of oxidative stress induced by exposure to organic solvents.