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

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

• BMB Reports
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• v.29 no.2
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• pp.111-115
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• 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 $45^{\circ}C$.

• The Korean Journal of Food And Nutrition
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• v.28 no.3
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• pp.458-464
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• 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
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• v.33 no.2
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• pp.179-183
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• 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.

• Korean Journal of Crystallography
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• v.14 no.1
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• pp.32-34
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• 2003

A helminth glutathione S-transferase. 28 kDa isozyme from Clonorchis sinensis has been crystallized under several conditions. One of the crystals, grown from a 10% polyehtylene glycol MME 550 (PEG MME 5 K) solution in 0.05 M potassium phosphate buffer (pH 7.0), diffracts to $3.0{\AA}$ resolution, and belongs to monoclinic space group C2, with unit cell parameters $a=95.83{\AA}$, $b=63.82{\AA}$, $c=235.09{\AA}$, and ${\beta}=97.2^{\circ}$.

• Korean Journal of Food Science and Technology
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• v.30 no.1
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• pp.206-212
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• 1998

This study was undertaken to explore the applicability of glutathione S-transferase (GST) activity as a predictable indicator to monitor chemical pollution in shells and fishes utilized for food. There were some variations in the basal level of GST activity depending on species tested. Ark shells, Anadara satowi, showed the highest normal enzyme activity, followed by catfish and marine mussels, Mytilus coruscus. White clams, Meretrix lusoria, Israeli carp and catfish had lower activity. When A. satowi was exposed to 3-methyl-cholanthrene (3-MC), a prototypic polycyclic aromatic hydrocarbon for 1 week, GST activity decreased by about 30%. This reduction in GST activity induced by 3-MC did not recover until two weeks after the cessation of exposure. GST activity increased in response to 3-MC in most of the other species studied. The GST elevation in M. coruscus attained its maxinum of about 200% at the termination of 3-MC exposure maintaining this level up to 2 weeks, and declined gradually thereafter. 3-MC also induced GST activity in lsraeli carp in a similar fashion to M. coruscus. Phenobarbital induced GST activity both in M. coruscus and lsraeil carp. Other chemicals. such as clofibrate, butylated hydroxyanisole. hexachlorobenzene, and oxolinic acid did not change the enzyme activity significantly in most speciel. Phenol depressed GST activity only in lsraeli carp. These results suggest that the basal level of GST activity is somewhat variable and that the direction of change in response to chemicals seems to be related to its normal activity. The change in enzyme activity can be a preditable indicator of some environmental chemicals such as PAHs and phenol.

• Korean Journal of Environmental Agriculture
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• v.16 no.1
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• pp.37-43
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• 1997

Tolerance mechanism to simazine (6-chloro-N,N'-diethyl-1,3,5-triazine-2,4-diamine) in Coix lacryma-jobi was investigated with respect to herbicide detoxification via glutathione conjugation. Simazine was initially absorbed by seedlings of C. lacryma-jobi and corn, but after 12 hours of treatment, no significant difference in simazine absorption was found in both species. Simazine absorbed was rapidly metabolized to glutathione-simazine conjugate. One to six hours after treatment, metabolism was approximately 2-fold faster in C. lacryma-jobi than in corn. Glutathione content was found 1.5- and 2.3-fold higher in coleoptile and root of C. lacryma-jobi, respectively, compared with corn. In both species, the highest concentration of glutathione was found in coleoptile tissue. Glutathione S-transferase that exhibits activity with 1-chloro-2,4-dinitrobenzene was not significantly different between two species. However, glutathione S-transferase activity with simazine was approximately 2-fold greater in C. lacryma-jobi than in corn. The glutathione S-transferase activity was 20 to 30% greater in shoot of either species than in root. Fast protein liquid chromatography-anion exchange column was used to separate glutathione S-transferase isozymes in coleoptiles of C. lacryma-jobi and corn. A peak of glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene and two peaks of glutathione S-transferase activity with simazine from C. lacryma-jobi were coeluted with those from corn, but showed greater activity than in the case of corn. Another glutathione S-transferase isozyme that exhibits activity with simazine was detected in the elution of C. lacryma-jobi extract, but not in corn. Electron transport in chloroplast thylakoids isolated from leaves of both species was equally sensitive to simazine applied at 1 to 100 nM. These results indicate that the simazine tolerance in C. lacryma-jobi is due to its capacity to detoxify the herbicide via glutathione conjugation, which is positively correlated with the level of glutathione content and glutathione S-transferase activity.

• The Korean Journal of Food And Nutrition
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• v.28 no.5
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• pp.866-870
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• 2015

In this study, coffee waste was extracted with different solvents such as ethyl acetate, methylene chloride and methanol to investigate the total polyphenol contents, electron donating ability and the inhibitory effect on glutathione S-transferase. The total polyphenol contents were $3,060.61{\pm}357.12{\mu}g\;GAE/mL$ in ethyl acetate, $909.09{\pm}35.71{\mu}g\;GAE/mL$ in methylene chloride, and $1,602.27{\pm}30.36{\mu}g\;GAE/mL$ in methanol. The total polyphenol contents showed a significant difference (p<0.05) between the solvents. The electron donating ability was $80.20{\pm}1.45%$ for ethyl acetate, $81.94{\pm}0.45%$ for methylene chloride, and $85.14{\pm}1.53%$ for methanol. The electron donating abilities were significantly different (p<0.05) between the solvents. The inhibitory effect of the various extracts on glutathione S-transferase (% inhibition) was $92.12{\pm}0.56%$, $88.48{\pm}0245%$ with methylene chloride extract, and $90.85{\pm}0.14%$ with methanol extract. These too were significant different (p<0.05) between the solvents. The two portions of coffee waste extracts obtained from ethyl acetate and methanol showed meaningful results on the total polyphenol contents, and the inhibition effects on glutathione S-transferase. Therefore, they can be utilized to develop health care foods and can be applied as antioxidants for cosmeceuticals.

• Journal of Plant Biotechnology
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• v.4 no.3
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• pp.117-122
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• 2002

Cotton Glutathione S-Transferase(GST: EC 2.5.1.18) was cloned and Gh-5 cDNA was overexpressed in tobacco (Nicotiana tabacum) plants. The transformation of cotton GST in tobacco plant was confirmed by northern blot analysis. Type I and Type II transcript patterns were identified in Gh-5 transgenic tobacco plants. Type I transcripts was only discussed in this paper. Glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) were used as the substrates, and the activity of GST in the type I transgenic plants was about 2.5-fold higher than the non-expressers and wild type tobacco plants. The expression of cotton GST in tobacco plants proved that Gh-5 could be translated into functional protein. Type I transgenic plants produced functional GST in the cells. Type I showed higher GST specific activity than Type II in the transgenic plants. Control and transgenic seedlings were grown in the growth chamber and under the light at 15$^{\circ}C$, and the effects of cotton GST in the seedlings was evaluated. The growth rate of Gh-5 overexpressors was better than the control and non-transgenic tobacco plants. Salinity tolerance was also analyzed on the seeds of transgenic plants. Seeds of Gh-5 overexpressors and the wild type tobacco seedlings were germinated and grown at 0, 50, 100, 150, and 200 mM NaCl solution. Gh-5 transgenic seedlings showed higher growth rate over control seedlings at both 50 and 100 mM NaCl solution. But at 0, 150, and 200 mM NaCl concentration, the difference in growth rate was not detected.