• The Korean Journal of Pesticide Science
• /
• v.7 no.1
• /
• pp.38-44
• /
• 2003

In vitro inhibitory activity of 34 insecticides and 31 fungicides to glutathione-S-transferase and esterases extracted from rats was determined. Of tested pesticides, the pesticides with high activity on both detoxifying enzymes were mixed with pesticides that are known to be detoxified by detoxifying enzymes. Glutathione-S-transferase was inhibited by thiodicarb $(I_{50}:1.87\times10^{-4}M)$, thiocyclam $(7.40\times10^{-4}M)$, dithianon $(7.55\times10^{-5}M)$, and tolylfluanide $(8.66\times10^{-5}M)$, while esterases by dichlorvos $(8.95\times10^{-8}M)$, pirimicarb $(2.74\times10^{-6}M)$, pyrazophos $(3.31\times10^{-5}M)$, and benomyl $(4.96\times10^{-5}M)$. After acephate known to be detoxified by glutathione-S-transferase was mixed with glutathione-S-transferase-inhibiting pesticides and phenthoate known to be detoxified by esterases was mixed with esterases-inhibiting pesticides, insecticidal activities of such mixtures were determined against diamondback moth (PlutelLa xylostella L.). Synergistic action was observed in all pesticide combinations. The highest synergistic action was obtained when phenthoate was combined with dichlorvos, showing that co-toxicity coefficients were 1512 and 1877 after 24 and 48 hours of treatment, respectively. Several other combinations of pesticides, such as phenthoate with benomyl, and acephate with dithianon, also showed synergism, showing that their co-toxicity coefficients were about 1,000 and 500, after 24 hours of treatment, respectively. Our results showed that combinations of pesticides inhibited by detoxifying enzymes and ones detoxified by detoxifying enzymes resulted in increased toxicities of pesticides, suggesting that such combinations could be used to develop pesticide mixtures with more broad spectrum and high effectiveness.

• The Korean Journal of Pesticide Science
• /
• v.6 no.1
• /
• pp.31-35
• /
• 2002

Objectives of this study were to determine if impurities in technical grade benfuracarb inhibit glutathione-S-transferase and amidase and to identify structures of impurities in technical grade benfuracarb. Technical grade benfuracarb, active ingredient, and impurity inhibited glutathione-S-transferase, and their $I_{50}$ were $9.7{\times}10^{-4}M,\;>1.0{\times}10^{-3}M,\;1.8{\times}10^{-4}M$, respectively. Such inhibition, however, was not higher than that by ethacrynic acid, a selective inhibitor to GST. Technical grade benfuracarb, active ingredient, and impurity also inhibited amidase, and their $I_{50}$ were $6.0{\times}10^{-5}M,\;4.3{\times}10^{-4}M,\;7.6{\times}10^{-5}M$, respectively. Our results show that the inhibition of both detoxifying enzymes by impurities in benfuracarb was 10-fold lower than that by active ingredient, suggesting that both active ingredient and impurities are involved in the inhibition of both detoxifying enzymes. Of four impurities (IM $1{\sim}4$) that were separated from technical grade benfuracarb, IM 2 and IM 3 inhibited GST and amidase. Based on data from IR, $^1H$-NMR, $^{13}C$-NMR and MS, it was determined that IM 2 is ethyl-N-isopropylamino propionate and IM 3 is ethyl-N-isopropyl-N(chlorosulfenyl)aminopropionate.

• BMB Reports
• /
• v.31 no.1
• /
• pp.77-82
• /
• 1998

To purify the geranylgeranyl protein transferase type I (GGPT-I) efficiently, a gene expression system using the pGEX-4T-1 vector was constructed. The cal1 gene, encoding the ${\beta}$ subunit of GGPT-I, was subcloned into the pGEX-4T-1 vector and co-transformed into E. coli cells harboring the ram2 gene, the ${\alpha}$ subunit gene of GGPT-I. GGPT-I was highly expressed as a fusion protein with glutathione S-transferase (GST) in E. coli, purified to homogeneity by glutathione-agarose affinity chromatography, and the GST moiety was excised by thrombin treatment. The purified yeast GGPT-I showed a dose-dependent increase in the transferase activity, and its apparent $K_m$ value for an undecapeptide fused with GST (GST-PEP) was $0.66\;{\mu}M$ and the apparent value for geranylgeranyl pyrophosphate (GGPP) was $0.071\;{\mu}M$.

• The Korean Journal of Pesticide Science
• /
• v.2 no.1
• /
• pp.97-103
• /
• 1998

Effects of safener fluxofenim was investigated for crop injury of acetanilide's upland herbicides and for enzyme activity of glutathione S-transferase (GST) in grain sorghum. Bioassay with etiolated grain sorghum [Sorghum bicolor (L.) Moench. cv. 'G522DR'] seedlings grown in agar containing metolachlor or alachlor showed that they are strong inhibitors on root growth of grain sorghum ($GI_{50}=4.5{\mu}M$ for metolachlor and $6.2{\mu}M$ for alachlor). The safener fluxofenim applied by seed soaking protected growth of grain sorghum from crop injury of metolachlor or alachlor at the concentrations of 1 to 10 ${\mu}M$. There was a significant increase in glutathione-herbicide conjugates in root tissues of fluxofenim-treated seedlings. Activities of $GST_{-metolachlor}$ and $GST_{-CDNB}$ were increased by 82% and 70%, respectively, in the cytosolic fraction of roots with fluxofenim treatment.

• BMB Reports
• /
• v.29 no.2
• /
• pp.111-115
• /
• 1996

A novel glutathione S-transferase from Pseudomonas sp. DJ77 was expressed in E. coli and purified by glutathione-affinity chromatography. The enzyme was composed of two identical subunits. The molecular size of the enzyme was 42 kDa by sephadex G-150 gel permeation chromatography and Mr of each subunit was 23 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. pI value of the enzyme was approximately 5.8 by isoelectric focusing. This enzyme showed the highest activity toward 1-chloro-2,4-dinitrobenzene as the electrophilic substrate. The relative activities toward p-nitrobenzyl chloride and 1,2-dichloro-4-nitrobenzene were 3.8% and 1.3% of the activity toward 1-chloro-2,4-dinitrobenzene, respectively. $K_m$ and $V_{max}$ values for 1-chloro-2,4-dinitrobenzene calculated by Lineweaver-Burk plot were 0.76 mM and $14.81\;{\mu}mol/min/mg$, respectively, and those for glutathione were 6.23 mM and $64.93\;{\mu}mol/min/mg$, respectively. The enzyme showed highest glutathione S-transferase activity at pH 8.0 and was stable between pH 6.0 and 9.0. The enzyme retained its activity up to $35^{\circ}C$ for 90 min but was unstable above $45^{\circ}C$.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.9 no.1
• /
• pp.65-71
• /
• 2004

A fat body-specific glutathione S-transferase cDMA was cloned from the spider, Araneus ventricosus. The cDNA encoding A. ventricosus glutathione S-transferase (AvGST) is 645 base pairs long with an open reading frame of 215 amino acid residues with a calculated molecular weight of approximately 24 kDa. Northern blot analysis showed the tissue-specifically expression of AvGST in the A. ventricosus fat body.

• The Korean Journal of Food And Nutrition
• /
• v.28 no.3
• /
• pp.458-464
• /
• 2015

In this study, the total polyphenol content, electron donating ability (EDA) and inhibitory activity of glutathione S-transferase (GST) of freeze-dried Citrus unshiu extracts were examined. The Citrus unshiu extracts was obtained from four solvents such as ethyl acetate, acetone, methyl chloride and methanol, to evaluate its functional properties. Total polyphenol contents were measured in the two different extracts, and the extracts were screened for their potential antioxidant activities using tests such as electron donating ability (EDA), glutathione S-transferase (GST). The total polyphenol contents of Citrus unshiu extracts were $928.48{\pm}1.19{\mu}g\;GAE/mL$ in ethyl acetate (EA), $886.03{\pm}0.44{\mu}g\;RE/mL$ in acetone (AC), $413.08{\pm}1.39{\mu}g\;GAE/mL$ in methylene chloride (MC), $12,648.60{\pm}0.56{\mu}g\;GAE/mL$ in methanol (MeOH), respectively. Also, the total polyphenol contents of EtOH Citrus unshiu extracts were $664.64{\pm}0.74{\mu}g\;GAE/mL$ in EA, $702.67{\pm}0.85{\mu}g\;RE/mL$ in AC, $429.64{\pm}0.61{\mu}g\;GAE/mL$ in MC, $16,108{\pm}0.73{\mu}g\;GAE/mL$ in MeOH, respectively. The total polyphenol contents were significantly difference (p<0.05) between the solvents. The electron donating ability of Citrus unshiu extracts were $62.80{\pm}0.36%$ in EA, $97.43{\pm}0.51%$ in AC, $52.20{\pm}0.30%$ in MC, $97.63{\pm}0.46%$ in MeOH, respectively. Also, the electron donating ability of EtOH Citrus unshiu extracts were $51.49{\pm}0.26%$ in EA, $63.17{\pm}0.31%$ in AC, $67.68{\pm}0.55%$ in MC, $96.18{\pm}0.41%$ in MA, respectively. The electron donating ability were significantly difference (p<0.05) between the solvents. The inhibitory activity of glutathione S-transferase in Citrus unshiu extracts were $76.22{\pm}0.65%$ in EA, $31.73{\pm}0.48%$ in MC, $97.48{\pm}0.56%$ in MeOH, respectively. Also, inhibitory activity of glutathione S-transferase in EtOH Citrus unshiu extracts were $75.54{\pm}0.55%$ in EA, $73.53{\pm}0.38%$ in MC, $48.70{\pm}0.46%$ in MeOH, respectively. The inhibitory activity of glutathione S-transferase were significantly difference (p<0.05) between the solvents. These results indicated that the Citrus unshiu extracts is a high-valued food ingredient and the extraction with methanol will be useful as a nutritional source with natural antioxidant activities. Considering high consumer demand beneficial health effects, Citrus unshiu extracts can be utilized to develop functional food health- promoting and natural antioxidant agents.

• BMB Reports
• /
• v.33 no.2
• /
• pp.179-183
• /
• 2000

The glutathione S-transferase (GST) activities of S-type and L-type thioltransferases (TTases), which are purified from the seeds and leaves of Arabidopsis thaliana, respectively, were identified and compared. The S-type and L-type TTases showed $K_m$ values of 9.72 mM and 3.18mM on 1-chloro-2,4-dinitrobenzene (CDNB), respectively, indicating the L-type TTase has higher affinity for CDNB. The GST activity of the L-type TTase was rapidly inactivated after being heated at $70^{\circ}C$ or higher. The GST activity of the S-type TTase remains active in a range of $30-90^{\circ}C$. $Hg^{2+}$ inhibited the GST activity of the S-type TTase, whereas $Ca^{2+}$ and $Cd^{2+}$ inhibited the GST activity of the L-type TTase. Our results suggest that the GST activities of two TTases of Arabidopsis thaliana may have different catalytic mechanisms. The importance of the co-existence of TTAse and GST activities in one protein remains to be elucidated.

• Bulletin of the Korean Chemical Society
• /
• v.24 no.8
• /
• pp.1188-1192
• /
• 2003

In order to study the role of residue in the active site of glutathione S-transferase (GST), Tyr 108 residue in human GST P1-1 was replaced with alanine, phenylalanine and tryptophan by site-directed mutagenesis to obtain mutants Y108A, Y108F and Y108W. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Tyr108 significantly affected $K_m^{CDNB}$ and $K_m^{ETA}$, whereas scarcely affected $K_m^{GSH}$. The substitutions of Tyr108 also significantly affected $I_{50}$ of ETA, an electrophilic substrate-like compound. The effect of these substitutions on kinetic parameters and the response to inhibition suggests that tyrosine 108 in hGST P1-1 contributes to the binding of the electrophilic substrate and a major determinant in the binding of CDNB is the aromatic ring of Tyr108, not its hydroxyl group.

• The Korean Journal of Pharmacology
• /
• v.32 no.2
• /
• pp.233-241
• /
• 1996

Previous studies have shown that glucocorticoid suppresses microsomal epoxide hydrolase(EH) gene expression and that EH expression is altered during pregnancy. The effects of progesterone on the expression of rat EH and certain glutathione S-transferase(GST) genes were examined in this study. Northern RNA blot analysis revealed that progesterone was effective in increasing hepatic EH mRNA levels at 12 h to 48 h after treatment with a maximal 9-fold increase being noted at 12 h time point. Nonetheless, multiple daily treatment with progesterone rather caused minimal relative increases in EH mRNA levels. GST Ya and Yb1/2 mRNA levels were also transiently elevated at 12 h after progesterone treatment, followed by gradual decreases from the maximal Increases at day 1, 2 and 5 post-treatment. These changes in EH and GST mRNA levels were noted only at a relatively high dose of progesterone. Furthermore, immunoblot analyses showed that rats treated with progesterone for 5 days failed to show EH or GST induction, indicating that progesterone-induced alterations in EH and GST mRNA levels do not reflect bona fide induction of the detoxifying enzymes. Concomitant progesterone treatment of rats with the known EH inducers including ketoconazole and clotrimazole failed to additively nor antagonistically alter EH mRNA levels. In contrast, dexamethasone substantially reduced ketoconazole- or clotrimazole-inducible EH expression. These results showed that progesterone stimulates the EH, GST Ya and Yb1/2 gene expression at early times followed by marked reduction in the RNA levels from the maximum after multiple treatment and that the changes in mRNA do not necessarily reflect induction of the proteins.