Properties Of Glutathione S-transferases Research Articles

Subcellular distribution and Nrf2/Keap1-interacting properties of Glutathione S-transferase P in hepatocellular carcinoma

The oncogene and drug metabolism enzyme glutathione S-transferase P (GSTP) is also a GSH-dependent chaperone of signal transduction and transcriptional proteins with key role in liver carcinogenesis. In this study, we explored this role of GSTP in hepatocellular carcinoma (HCC) investigating the possible interaction of this protein with one of its transcription factor and metronome of the cancer cell redox, namely the nuclear factor erythroid 2–related factor 2 (Nrf2).Expression, cellular distribution, and function as glutathionylation factor of GSTP1-1 isoform were investigated in the mouse model of N-nitrosodiethylamine (DEN)-induced HCC and in vitro in human HCC cell lines. The physical and functional interaction of GSTP protein with Nrf2 and Keap1 were investigated by immunoprecipitation and gene manipulation experiments.GSTP protein increased its liver expression, enzymatic activity and nuclear levels during DEN-induced tumor development in mice; protein glutathionylation (PSSG) was increased in the tumor masses. Higher levels and a preferential nuclear localization of GSTP protein were also observed in HepG2 and Huh-7 hepatocarcinoma cells compared to HepaRG non-cancerous cells, along with increased basal and Ebselen-stimulated levels of free GSH and PSSG. GSTP activity inhibition with the GSH analogue EZT induced apoptotic cell death in HCC cells. Hepatic Nrf2 and c-Jun, two transcription factors involved in GSTP expression and GSH biosynthesis, were induced in DEN-HCC compared to control animals; the Nrf2 inhibitory proteins Keap1 and β−TrCP also increased and oligomeric forms of GSTP co-immunoprecipitated with both Nrf2 and Keap1. Nrf2 nuclear translocation and β−TrCP expression also increased in HCC cells, and GSTP transfection in HepaRG cells induced Nrf2 activation.In conclusion, GSTP expression and subcellular distribution are modified in HCC cells and apparently contribute to the GSH-dependent reprogramming of the cellular redox in this type of cancer directly influencing the transcriptional system Nrf2/Keap1.

Effects of conserved Arg20, Glu74 and Asp77 on the structure and function of a tau class glutathione S-transferase in rice.

The relative position of domains is critical for enzymatic properties of tau class glutathione S-transferases, and altering the position of linker far away from the active center affects catalytic property. Glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes whose main function is to improve plant resistance to stresses. To understand the structural effects of tau class GSTs on their function, using OsGSTU17 as an example, we predicted the residues involved in the interactions between its domains and linker region. We further detected the structural changes in mutants and the corresponding changes in terms of substrate activity and kinetic parameters. Four pairs of residues, including Ala14 and Trp165, Arg20 and Tyr154, Glu74 and Arg98, Asp77 and Met87, forming hydrogen bonds and salt bridges were found to play important roles in maintaining the relative position between the domains and linker region inside the protein. The hydrogen bond between Trp165 and Ala14 affected the structural stability has been demonstrated in our previous study. The mutant R20A lost almost all catalytic activity. Interestingly, the mutant E74A exhibited a significant decrease in activity towards 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole, 1-chloro-2, 4-dinitrobenzene and 4-nitrobenzyl chloride, while its activity towards substrate cumene hydroperoxide remained unchanged. Compared with other mutants, the mutant D77A exhibited decreased affinity to its substrates and increased activity towards 1-chloro-2, 4-dinitrobenzene and cumene hydroperoxide, but its thermodynamic stability did not change significantly. The relative position of individual domain was critical for enzymatic properties, and the linker which is far away from the active site could change the enzymatic properties of GSTs via altering the relative position of the individual domain. Our results provide insights into the relationship between structure and function of tau class GSTs.

Alteration of enzyme activities and kinetic properties of GST and NQO1 with naturally occurring phenolic compounds

Abstract Objective: Glutathione S-transferase (GST) and NAD(P)H:quinine oxidoreductase 1 (NQO1) are the enzymes important in cytoprotection and bioactivation of chemicals. This study has addressed effects of polyphenolic compounds; ellagic acid, quercetin, naringenin, resveratrol, rutin and hesperidin on rabbit liver GST and NQO1 enzyme activities. Methods: Cytosolic fractions were obtained from homogenized liver tissues of rabbit by differential centrifugation. After calculating IC50 values of individual enzymes for phenolic compounds, both Lineweaver-Burk and Dixon plots were drawn to determine the effect of phenolics on enzyme activity. Michaelis-Menten constant (Km), maximum velocity (Vmax), and inhibition constant (Ki) were calculated from Lineweaver-Burk and Dixon plots, respectively. Results: Resveratrol was found to be the most potent inhibitor for rabbit hepatic cytosolic GST activity with 75.9±2.06 μM IC50 value while naringenin was the least potent one with IC50 value of 260±1.92 μM. Hesperidin and quercetin were found to be the most and least potent inhibitors for NQO1 enzyme activity with IC50 values of 2.7±0.85 μM and 13.8±0.91 μM, respectively. Resveratrol and naringenin inhibited GST activity noncompetitively and mixed type with Ki of 6.2 μM and 245 μM, respectively; while both hesperidin with 0.64 μM Ki value and quercetin with 3.5 μM Ki value inhibited NQO1 activity in a competitive manner. Conclusion: These results indicate that phenolic compounds may modulate Phase II enzyme, GST and NQO1. Moreover, they can influence the metabolic activation of xenobiotic and toxic compounds metabolized by this enzyme.

Purification and properties of glutathione S-transferase from the pike liver

Purification and properties of glutathione S-transferase from the pike liver

Characterization of glutathione S-transferase of the rice leaffolder moth, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae): Comparison of its properties of glutathione S-transferases from other lepidopteran insects

An enzyme that possesses the glutathione S-transferase (GST) activity was found in the rice leaffolder moth, Cnaphalocrocis medinalis. The enzyme was purified to homogeneity for the first time by ammonium sulfate fractionation and affinity chromatography. The resultant enzyme revealed a single band with a molecular mass of 24 kDa by SDS–polyacrylamide gel electrophoresis under reduced conditions. When assayed with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, the purified GST had an optimum pH at 8.0, and was fairly stable at pH 3–10 and at temperatures below 50 °C. The enzyme was also able to conjugate glutathione to 4-hydroxynonenal, a cytotoxic lipid peroxidation product. The present GST was inhibited by fenitrothion, permethrin, and deltamethrin, suggesting that the GST could be involved in metabolizing these organophosphorus and pyrethroid insecticides.

In silico identification of silkworm selenoproteomes

Selenium (Se) is an essential trace element in vivo. Its biological function is mainly exerted through selenoproteins. Selenocysteine (Sec), the active site of selenoproteins, is incorporated into the protein at an in-frame TGA codon under the guidance of Sec insertion sequence (SECIS) element in the 3′-untranslated region (UTR) of the gene. In this work, a method was developed and a series of programs were edited by PERL language to in silico identify selenoproteomes from the genome of domesticated silkworm (Bombyx mori). Out of 18510 annotated genes, 6348 was terminated with TGA codons, 249 containing both in-frame TGAs and SECIS elements in the 3′-UTRs. Alignments of those selenoprotein candidates with their cysteine (Cys)-containing homologs revealed that 52 genes had TGA/Cys pairs and similar flanking regions around the in-frame TGAs. Restricted by the pattems of SECIS elements only 5 genes were screened out to fully meet the requirements for selenoproteins. Among them glutathione S-transferase (GST) has been reported as a microbial selenoprotein, the other four are novel selenoproteins annotated as CG6024, CG5195, ATP-binding cassette transporter subfamily A (ABCA), and nuclear VCP-like protein. Derived from the general properties of GST, ABCA and VCP, silkworm selenoproteins may play important roles in redox regulation, Se storage and transportation, as well as cell apoptosis.

Occurrence and properties of glutathione S-transferases in phenol-degrading Pseudomonas strains

Pseudomonas sp. strains, able to degrade aromatic compounds such as phenol, were chosen to investigate the occurrence and characteristics of glutathione S-transferases (GSTs). Affinity chromatography purification showed the presence of at least one GST in each studied strain. The purified proteins exhibited a great variety in the N-terminal sequences and different enzyme activities with the standard GST substrates tested. Two Pseudomonas strains, M1 and CF600, were chosen to investigate the GST activities under different growth conditions. Therefore, cells were grown either on phenol or on different nonaromatic carbon sources in the presence and absence of increasing phenol concentrations. In strain M1 a strong correlation between the activities of the catechol 1,2-dioxygenase and GST was observed in all the tested conditions. Moreover, growth on different organic acids also affected GST activity levels, with a negative correlation with the specific growth rate determined by each substrate. These results suggest a possible function of GST as a response to specific metabolic conditions determined by phenol toxicity and/or catabolism and the metabolic status of the cells. The same experiments performed with the CF600 strain did not show induction of GST activity in any of the tested conditions, indicating that GST_CF600 probably has a different role in cell metabolism. Native gel electrophoresis gave indications that GST dimerization could be an important process in the modulation of GST activity.

PLASMA GLUTATHIONE S-TRANSFERASE π1-1 AND α1-1 LEVELS IN PATIENTS WITH BLADDER CANCER

No AccessJournal of UrologyINVESTIGATIVE UROLOGY1 Dec 2000PLASMA GLUTATHIONE S-TRANSFERASE π1-1 AND α1-1 LEVELS IN PATIENTS WITH BLADDER CANCER CHRIS L. BERENDSEN, THEO P.J. MULDER, and WILBERT H.M. PETERS CHRIS L. BERENDSENCHRIS L. BERENDSEN More articles by this author , THEO P.J. MULDERTHEO P.J. MULDER More articles by this author , and WILBERT H.M. PETERSWILBERT H.M. PETERS More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(05)66982-0AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: Transitional cell carcinomas of the human bladder and many gastrointestinal tumors often contain high amounts of the detoxification enzyme glutathione S-transferase π (GSTP1-1). Elevated levels of GSTP1-1 have been found in serum and plasma from patients with gastrointestinal, lung or head and neck cancer. GSTP1-1 and glutathione S-transferase α (GSTA1-1) have been reported to be increased in 10 of 15 patients (67%) with bladder cancer. We evaluate the role of GSTP1-1 and GSTA1-1 as plasma tumor markers in 50 patients with bladder cancer before and after treatment. Materials and Methods: Blood from patients with bladder cancer was sampled in ethylenediaminetetraacetic acid tubes. Plasma GSTA1-1 and GSTP1-1 were measured using the sensitive and specific sandwich enzyme-linked immunosorbent assay. Results: Respective plasma GSTA1-1 and GSTP1-1 levels were above the upper normal reference limit in 2 (4%) and 14 (28%) of the 50 patients with bladder cancer. No significant decrease in plasma GSTA1-1 or GSTP1-1 was noted in matched pairs of plasma samples collected before and after treatment. Conclusions: In contrast to earlier reports, only a limited number of patients with bladder cancer had increased plasma GSTA1-1 or GSTP1-1, which did not decrease after tumor resection. These findings argue against the use of GSTP1-1 or GSTA1-1 as plasma markers for bladder cancer. References 1 : The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol1995; 30: 445. Google Scholar 2 : Nomenclature for human glutathione transferases. Biochem J1992; 282: 305. Google Scholar 3 : Preparation, characterization and properties of glutathione S-transferases. In: . Edited by . New York: Wiley J and Sons1985: 297. Google Scholar 4 : The isoenzymes of glutathione S-transferase. In: Advances in Enzymology. Edited by . New York: Wiley J and Sons1985: 357. Google Scholar 5 : Glutathione S-transferases: biomedical applications. Adv Clin Chem1993; 30: 282. Google Scholar 6 : Glutathione transferases and cancer. Crit Rev Biochem Mol Biol1992; 27: 337. Google Scholar 7 : Glutathione S-transferase activity and subunit composition in transitional cell cancer and mucosa of the human bladder. Urology1997; 49: 644. Google Scholar 8 : Enhanced glutathione S-transferase activity and glutathione content in human bladder cancer. Follow up study: influence of smoking. J Urol1993; 149: 1452. Google Scholar 9 : Glutathione S-transferase activity in human superficial transitional cell carcinoma of the bladder. Cancer1990; 9: 2064. Google Scholar 10 : Enzyme immunoassay for human serum glutathione S-transferase pi using monoclonal and polyclonal antibodies. Cancer J1989; 2: 225. Google Scholar 11 : Serum glutathione S-transferase-pi as a tumor marker for gastrointestinal malignancies. Cancer1989; 63: 317. Google Scholar 12 : Elevation of the placental glutathione S-transferase form (GST-pi) in tumor tissues and the level in sera of patients with cancer. Cancer Res1989; 49: 5225. Google Scholar 13 : Serum glutathione S-transferase-pi level as a tumor marker for non-small cell lung cancer. Cancer1994; 73: 1377. Google Scholar 14 : Radioimmunoassay for plasma glutathione S-transferase-pi and its clinical application in gastrointestinal cancer. Cancer1995; 76: 1363. Google Scholar 15 : Significance of glutathione S-transferase-pi as a tumor marker in patients with oral cancer. Cancer1992; 70: 2381. Google Scholar 16 : Isoenzyme-specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines. Biochim Biophys Acta1994; 1225: 223. Google Scholar 17 : Sandwich ELISA for glutathione S-transferase Alpha 1-1: plasma concentrations in controls and in patients with gastrointestinal disorders. Clin Chem1996; 42: 416. Google Scholar 18 : Measurement of glutathione S-transferase P1–1 in plasma; pitfalls and significance for screening and follow-up of patients with gastrointestinal cancer. Cancer1997; 80: 873. Google Scholar 19 : Hepatocellular integrity in liver donors and recipients indicated by glutathione transferase alpha. Transplantation1996; 61: 904. Google Scholar 20 : Glutathione S-transferase alpha as a marker for hepatocellular damage in pre-eclampsia and HELLP syndrome. Lancet1995; 345: 1571. Google Scholar From the Department of Urology, Atrium Medical Center Heerlen, Heerlen and Department of Gastroenterology, University Hospital St. Radboud, Nijmegen, The Netherlands© 2000 by American Urological Association, Inc.FiguresReferencesRelatedDetails Volume 164Issue 6December 2000Page: 2126-2128 Advertisement Copyright & Permissions© 2000 by American Urological Association, Inc.Keywordsglutathione transferasetumor markers, biologicalenzyme-linked immunosorbent assaybladder neoplasmsMetricsAuthor Information CHRIS L. BERENDSEN More articles by this author THEO P.J. MULDER More articles by this author WILBERT H.M. PETERS More articles by this author Expand All Advertisement PDF downloadLoading ...

Purification and some properties of glutathione S-transferase from Escherichia coli B

Glutathione S-transferase was purified approximately 2,300-fold from cell extracts of Escherichia coli B with a 7.5% activity yield. The molecular weight of the enzyme was 45,000, and the enzyme appeared to consist of two homogeneous subunits. The enzyme was almost specific to 1-chloro-2,4-dinitrobenzene (Km, 1.43 mM) and glutathione (Km, 0.33 mM). The optimal pH and optimal temperature for activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable from pH 5 to 11. The activity of the enzyme for 1-chloro-2,4-dinitrobenzene (3,2 mumol/min per mg of protein) was significantly lower than those of the enzymes from mammals, plants, and fungi.

Purification and characterization of multiple forms of rabbit hepatic glutathione S-transferase.

Multiple forms and properties of glutathione S-transferase were investigated by using the liver from individual male or female rabbits. Most of the activity toward 1-chloro-2, 4-dinitrobenzene (CDNB) in the crude extract passed through a diethylaminoethyl (DEAE)-cellulose column. On carboxymethyl (CM)-cellulose chromatography, the flow-through fraction was resolved into at least four activity peaks which were designated as R1, R2, R3 and R4 in order of elution. The content of each component varied among the individual rabbit livers examined. The main components, R2 and R3, were further purified to homogeneity as judged by sodium deodecyl sulfate (SDS)/polyacrylamide-gel electrophoresis. R3 contained two forms, whcih were named R3a and R3b in order of elution from a hydroxylapatite column. R2, R3a and R3b each had a molecular weight of approximately 51000 as estimated by gel filtration. R2 and R3b appeared to be homodimers consisting of subunits of apparently different size (R2, Y2 : 25000; R3b, Y3 : 2650). On the other hand, R3a was a heterodimer composed of subunits with molecular weights of 24500 (Y1) and 26500 (Y3). The molecular weight of the subunit of R3b was identical with that of Y3. These enzymes showed the highest activity toward CDNB and much lower activities toward the other substrates tested, and the substrate specificities of all the enzymes were roughly similar to each other.Our highly purified enzyme forms were compared with rat hepatic glutathione S-transferases with regard to substrate specificity, subunit species, kinetic parameters, isoelectric point and amino acid composition.

Distribution and properties of glutathione S-transferase from T. infestans

1. 1. The glutathione transferase from T. infestans is able to render aqueous metabolites when incubated in vitro with malathion, parathion and fenitrothion. 2. 2. It is a soluble enzyme present in every developmental stage and widely distributed in all insect organs. 3. 3. The purification procedure applied, consisting of fractionation with ammonium sulfate and Bio-Gel P-60 chromatography, gives an unique molecular form catalytically active using methyl iodide as substrate in polyacrylamide gel electrophoresis (PAGE). 4. 4. One of the most active substrates is the 1-chloro-2,4-dinitrobenzene (CDNB), with an activity maximum at pH 7.5 and at 45°C temperature. Its activation energy calculated from an Arrhenius plot is 14,846 cal mol −1. 5. 5. The enzyme susceptibility to inhibition by thiol reagents shows three degrees of responses; slight, moderate or high, depending on the compounds used. 6. 6. The kinetics of the enzyme catalysed reaction with the purified fraction is complex, and resembles that reported for glutathione S-transferase A from rat liver, showing a biphasic kinetic mechanism in which the reaction pathway depends on the concentration of GSH. 7. 7. In general, the properties of this insect enzyme are similar to those enzymes isolated from vertebrate organisms.

The binding and conjugating abilities of the Y-fraction for sulfobromophthalein (BSP) in chronic carbon tetrachloride-intoxicated rats.

It was revealed by the Sephadex G-75 gel filtration of rat liver supernatant containing sulfobromophthalein (BSP) that the rats chronically intoxicated with CCl4 have lower BSP conjugating activities as well as lower protein contents in the Y-fraction than the control rats. Enzyme kinetic studies on the Y-fraction were carried out in order to investigate the changes induced by intoxication in the properties of glutathione S-transferases in the Y-fraction with BSP as a substrate. Since neither Km (Michaelis-Menten constant) nor Vmax (maximum velocity) value changed, no alteration by the intoxication was observed in the Y-fraction as a conjugating enzyme. It is clear that the intoxication brought about a decrease in the enzyme content in the Y-fraction, considering that protein concentration of the Y-fraction on the gel filtration decreased without the change of Vmax value. Our previous report showed that the content of endogeneous glutathione (GSH) was not altered by the intoxication. These observation led to the conclusion that the decreased BSP conjugating activity by the intoxication was due to the decreased content of enzyme in the Y-fraction. The BSP binding to the Y-fraction was measured by equilibrium dialysis. It was found that the intoxication reduced the binding constant by more than 50%, whereas the change of dissociation constant (Km) estimated from the enzyme kinetics was not observed.