Glutathione-agarose Affinity Chromatography Research Articles

Glutatyon S-Transferaz Enziminin Tavuk Karaciğerinden Saflaştırılması ve Karakterizasyonu

In this study, the glutathione S-transferase enzyme (GST; EC 2.5.1.18) was purified with 8.35 EU/mL specific activity, 24.56 times 8% yield, from chicken liver, using ammonium sulfate precipitation and glutathione-agarose affinity chromatography. In order to control the purity of the enzyme, SDS-PAGE was performed and a single band was obtained. The molecular mass of the subunit was calculated as approximately 30.9 kDa. In addition, the optimum pH value of the enzyme (8.5 in Tris-HCl); optimum ionic strength (150 mM with Tris-HCl); optimum temperature (70 oC); stable pH value (8.5 with Tris-HCl) was determined. The KM value for the GSH substrate of the enzyme was 0.802 mM, the Vmax value was 1.833 EU/mL; For CDNB, the KM value was calculated as 3.6 mM and the Vmax value was calculated as 2.829 EU/mL.

In vitro and in silico enzyme inhibition effects of some metal ions and compounds on glutathione S-transferase enzyme purified from Vaccinium arctostapylous L.

Glutathione s-transferase (GST) is a class of enzymes that performs a wide array of biological functions. However, GST enzymes are most famously known for their roles in catalyzing the conjugation of reduced glutathione (GSH) to electrophilic centers on a wide variety of substrates to induce water-solubility to compounds as a protective antioxidant mechanism against toxic substances. In the present study, in vitro inhibition effects of coumarin, ascorbic acid, sodium sulfide, sodium azide, citric acid compounds, and Cd2+, Cu2+, Ni2+, Mg2+ metal ions against GST enzyme were determined. For this aim, the GST enzyme was purified from Vaccinium arctostapylous L. using the glutathione-agarose affinity chromatography and Sephadex G-100 gel filtration steps. The respective metals and chemical compounds were used at different concentrations for measuring their in vitro GST activity effects. The Ki values of these agents were determined as 0.450 ± 0.13, 15.05 ± 7.05, 0.009 ± 0.001, 0.022 ± 0.006, 0.120 ± 0.36, 0.150 ± 0.06, 0.223 ± 0.03, 0.002 ± 0.0003, and 0.136 ± 0.06 mM, respectively. Finally, the molecular docking interactions of the compounds with the GST target enzyme were evaluated using Autodock Tools-1.5.6. The effective molecular interactions of coumarin, citric acid, ascorbic acid, and sodium sulfide with GST target enzyme were found with their binding lowest energy affinities −4.62, −3.04, −2.53, and −1.67 kcal/mol, respectively. Communicated by Ramaswamy H. Sarma

Glutathione S-transferase: Purification and Characterization from Quail (Coturnix coturnix japonica) Liver and the Impact of Some Metal Ions on Enzyme Activity

The objective of this study was to examine the inhibitory effect of some heavy metals on the glutathione S-transferase (GST) enzyme, which is one of the major enzymes of glutathione metabolism purified from quail liver tissues. The quail liver GST enzyme was purified with 15.86 EU/mg specific activity, in a yield of 12.36% and 46.1 purification fold by ammonium sulfate precipitation and glutathione-agarose affinity chromatography. The molecular weight of subunits of the enzyme and the purity were determined by SDS-PAGE. In the characterization studies, the optimum pH of the GST enzyme was determined to be pH = 8.0 in Tris/HCl buffer. Optimum ionic strength was determined to be 140 mM in Tris/HCI buffer. Stable pH was found to be pH = 8.5 in Tris/HCl buffer. Optimum temperature was found to be 50 °C. KM and Vmax values for substrates 1-chloro-2,4-dinitrobenzene (CDNB) and GSH of the enzyme were determined to be KM 0.048 mM Vmax 0.479 EU/mL and KM 0.114 mM Vmax 0.672 EU/mL, respectively. In vitro inhibition effects of metal ions, including Ag+, Ni2+, Cd2+, Fe2+, Pb2+, Co2+ Zn2+, and Al3+, were investigated on the GST enzyme activity. The results showed that Ag+, Cd2+, Ni2+ Zn2+, and Al3+ metal ions inhibited GST enzyme (IC50 values 0.239, 0.250, 0.265, 0.320, 0.594 mM, respectively), while Fe2+, Pb2+, and Co2+ metal ions activated the enzyme. Finally, Ki values and inhibition types for these substances were determined by Lineweaver-Burk graphs.

Deciphering of The Effect of Chemotherapeutic Agents on Human Glutathione S-Transferase Enzyme and MCF-7 Cell Line.

Cancer is the disease that causes the most death after cardiovascular diseases all over the world these days. Breast cancer is the most common type of cancer among women and ranks the second among cancer-related deaths after lung cancer. Chemotherapeutics act by killing cancer cells, preventing their spread and slowing their growth. Recent studies focus on the effects of chemotherapeutics on cancer cells and new chemotherapy approaches that targeting enzymes that catalyze important metabolic reactions in the cell. The aim of this study was to investigate the effects of chemotherapeutic agents, Tamoxifen and 5-FU, on MCF-7 cell line and human erythrocyte GST, an important enzyme of intracellular antioxidant metabolism. In this study, it was investigated that the effect of chemotherapeutic agents, Tamoxifen and 5-FU, on MCF-7 breast cancer cell line and performed ROS analyzes. In addition, it was purified glutathione S-transferase (GST), one of the important enzymes of intracellular antioxidant mechanism, from human erythrocytes by using ammonium sulfate precipitation and glutathione agarose affinity chromatography, and investigated in vitro effects of chemotherapeutic agents, 5 - FU and Tamoxifen, on the activity of this enzyme for the first time. it was determined that Tamoxifen and 5-FU inhibited cellular viability and 5-FU increased intracellular levels of ROS, whereas Tamoxifen reduced intracellular levels of ROS. In addition, human erythrocyte GST enzyme with 16.2 EU/mg specific activity was purified 265.97-fold with a yield of 35% using ammonium sulfate precipitation and glutathione agarose affinity chromatography. The purity of the enzyme was checked by the SDS-PAGE method. In vitro effects of chemotherapeutics, 5-FU and Tamoxifen, on GST activity purified from human erythrocytes were investigated. The results showed that 5-FU increased the activity of GST in the concentration range of 77 to 1155 μM and that Tamoxifen increased the activity of GST in the concentration range of 0.54 to 2.70 μM. In this study, the effects of tamoxifen and 5-FU chemotherapeutic agents on both MCF-7 cell line and human GST enzyme were examined together for the first time. Our study showed that chemotherapeutic agents (5-FU and Tamoxifen) inhibited cellular viability and Tamoxifen reduced intracellular levels of ROS whereas 5-FU increased intracellular levels of ROS. In addition, 5-FU and Tamoxifen were found to increase the activity of GST enzyme purified from the human erythrocyte.

The interaction methylene blue and glutathione-S-transferase purified from human erythrocytes

It is known that textile dyes have various risks on human health. Glutathione-S-transferase enzymes play a critical role in the detoxification of xenobiotics in living systems. This study aimed to examine the interaction of methylene blue with human erythrocyte glutathione-S-transferase purified in one step. Human erythrocyte glutathione-S-transferase was purified with approximately 750-fold purification and 30% efficiency by glutathione agarose affinity chromatography. The results showed that the enzyme was inhibited by methylene blue with an IC50 value of 1.40 mmol/L. The Ki constant of methylene blue was 1.17 mmol/L. The Lineweaver–Burk graph of the methylene blue showed that the type of inhibition was compatible with mixed type inhibition. A new third spot was also detected by thin-layer chromatography. Furthermore, the cytotoxicity of methylene blue on human erythrocytes was evaluated and it was found that the haemolysis per cent of methylene blue on erythrocytes was approximately 14%.

Purification of native Sigma class glutathione transferase from Fasciola hepatica

Fascioliasis is a parasitic disease of grazing livestock and a threat to global food security by significantly reducing the production value of sheep, goats and cattle. Moreover, the zoonotic parasite is also a re-emerging food borne threat to human populations. Driven by climate change, the prevalence of fascioliasis is set to increase. Efforts to control the main causative agent, Fasciola hepatica, are hampered by short lived chemotherapy approaches that are becoming increasingly obsolete due to therapeutic failure and resistance. A protective vaccine is urgently needed. A recombinant form of Sigma class glutathione transferase (Hematopoietic Prostaglandin D synthase) from F. hepatica (FhGSTS1) with confirmed prostaglandin synthase activity shows immune-modulation activity via suppression of Th17 responses in host dendritic cells. In vaccine trials recombinant FhGSTS1 reduces liver pathology but not worm burden. Native FhGSTS1 is yet to be tested for immune-modulation activities or for vaccine potential, primarily due to the technical difficulty in purifying FhGST-S1 away from the other more abundant GST members in F. hepatica cytosol. This paper reports a pipeline for the purification of native FhGSTS1 using a two-step process consisting of glutathione-agarose affinity and cationic exchange chromatography. The methodology allows for the isolation of purified and active FhGSTS1 or Sigma GSTs from other sources for analytical biochemical and immunological studies.

Purification of glutathione S-transferase enzyme from quail liver tissue and inhibition effects of (3aR,4S,7R,7aS)-2-(4-((E)-3-(aryl)acryloyl)phenyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione derivatives on the enzyme activity.

The use of quail meat and eggs has made this animal important in recent years, with its low cost and high yields. Glutathione S-transferases (GST, E.C.2.5.1.18) are an important enzyme family, which play a critical role in detoxification system. In our study, GST was purified from quail liver tissue with 47.88-fold purification and 12.33% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by SDS-PAGE method and showed a single band. In addition, inhibition effects of (3aR,4S,7R,7aS)-2-(4-((E)-3-(aryl)acryloyl)phenyl)-3a,4,7,7a-tetrahydro-1H-4,7methanoisoindole-1,3(2H)-dion derivatives (1a-g) were investigated on the enzyme activity. The inhibition parameters (IC50 and Ki values) were calculated for these compounds. IC50 values of these derivatives (1a-e) were found as 23.00, 15.75, 115.50, 10.00, and 28.75μM, respectively. Ki values of these derivatives (1a-e) were calculated in the range of 3.04±0.50 to 131.50±32.50μM. However, for f and g compounds, the inhibition effects on the enzyme were not found.

Inhibition of human erythrocyte glutathione S-transferase by some flavonoid derivatives

Glutathione transferase is one of the important enzymes for xenobiotic metabolism. Glutathione transferases catalyze the conjugation of various electrophilic endogenous and exogenous xenobiotics with γ-glutamyl-cysteinyl-glycine (GSH). GST activity can be changed by natural plant products. The present study’s goal is to examine the interaction of human erythrocyte GST with the natural plant compounds baicalin, baicalein, phloridzin, and phloretin. First, GST was purified from human erythrocyte with 1654-fold purification and 19.27% recovery by glutathione agarose affinity chromatography. The purity of the enzyme was checked by SDS-PAGE, showing single band, because it had a homodimer structure. Baicalin, baicalein, phloridzin, and phloretin flavonoids were shown to inhibit human erythrocyte GST with the IC50 values of 28.75, 57.50, 769.10, and 99.02 μM, respectively. The Ki constants for baicalin, baicalein, phloridzin, and phloretin flavonoids were 14.50 ± 0.71, 24.33 ± 2.08, 762.50 ± 85.97, and 86.49 ± 1.11 μM, respectively. According to these results, baicalin is the best inhibitor for human erythrocyte GST.

Characterization and Purification of Glutathione S-Transferase from the Liver and Gill Tissues of Ağrı Balık Lake Trout Salmo trutta labrax and the Effects of Heavy Metal Ions on Its Activity.

Glutathione S-transferase (GST) from the liver and gill tissues of Ağrı Balık Lake Trout (also known as Black Sea Trout) Salmo trutta labrax was characterized and purified, and the toxic effects of some heavy metal ions on the enzyme's activity were analyzed. Liver GST was purified 930 times, resulting in 56% yield using glutathione-agarose affinity chromatography and a specific activity of 60.87 endotoxin units (EU)/mg protein. Using the same procedure, gill GST was purified 576 times, resulting in a 60% yield and specific activity of 46.8 EU/mg protein. The purity check of the purified enzymes was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal pH, ionic strength, and stable pH were found for each tissue, and separate KM and Vmax values were determined for reduced glutathione and 2,4-dinitrochlorobenzene substrates. Heavy metal ions that have toxic effects on living organisms and are known to contribute to environmental pollution were selected, and their in vitro effects on enzyme activity were analyzed. The IC50 values and Ki constants of those metal ions showing an inhibitory effect on GST activity were determined.

Glutathione Transferases Responses Induced by Microcystin-LR in the Gills and Hepatopancreas of the Clam Venerupis philippinarum.

A multi-method approach was employed to compare the responses of Glutatione Transferases (GSTs) in the gills and hepatopancreas of Venerupis philippinarum to microcystins (MCs) toxicity. In this way, using the cytosolic fraction, the enzymatic activity of GSTs, superoxide dismutase (SOD), serine/threonine protein phosphatases (PPP2) along with the gene expression levels of four GST isoforms (pi, mu, sigma1, sigma2) were investigated in both organs of the clams exposed for 24 h to 10, 50 and 100 μg L−1 of MC-LR. Cytosolic GSTs (cGSTs) from both organs of the high dose exposed clams were purified by glutathione-agarose affinity chromatography, characterized kinetically and the changes in the expression of cGSTs of the gills identified using a proteomic approach. MC-LR caused an increase in GST enzyme activity, involved in conjugation reactions, in both gills and hepatopancreas (100 μg L−1 exposure). SOD activity, an indicator of oxidative stress, showed significantly elevated levels in the hepatopancreas only (50 and 100 μg L−1 exposure). No significant changes were found in PPP2 activity, the main target of MCs, for both organs. Transcription responses revealed an up-regulation of sigma2 in the hepatopancreas at the high dose, but no significant changes were detected in the gills. Kinetic analysis evidenced differences between gills of exposed and non-exposed extracts. Using proteomics, qualitative and quantitative differences were found between the basal and inducible cGSTs. Overall, results suggest a distinct role of GST system in counteracting MCs toxicity between the gills and the hepatopancreas of V. philippinarum, revealing different roles between GST isoforms within and among both organs.

Purification of glutathione S-transferase from Van Lake fish (Chalcalburnus tarichii Pallas) muscle and investigation of some metal ions effect on enzyme activity

Glutathione S-transferases (GSTs) are an important enzyme family which play a critical role in detoxification system. In our study, GST was purified from muscle tissue of Chalcalburnus tarichii Pallas with 301.5-fold purification and 19.07% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, showing a two band, because of having heterodimer structure. KM values were 1.59 and 0.53 mM for 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), respectively. Vmax values for CDNB and GSH were also determined as 5.58 and 1.88 EU/mL, respectively. In addition, inhibition effects of Ag+, Cu2+, Cd2+, Fe3+, Pb2+, Cr2+, Co2+ and Zn2+ metal ions were investigated on the enzyme activity and IC50, Ki values were calculated for these metal ions.

Expression, purification, and therapeutic implications of recombinant sFRP1.

Secreted frizzled-related proteins (sFRPs) constitute a family of proteins, which impede the Wnt signaling pathway. Upregulation of the Wnt cascade is one of the multiple facets of carcinogenesis. Herein, we report the expression, solubilization, purification, characterization, and anti-cell proliferative activity of a novel recombinant GST-tagged sFRP1 of human origin. sFRP1 was cloned into pGEX-4T2 bacterial expression vector, and the recombinant protein was overexpressed in Escherichia coli BL21 (DE3). It was solubilized from inclusion bodies with N-lauroylsarcosine and Triton X-100, before being purified to homogeneity using glutathione agarose affinity chromatography column. The purified protein was characterized using Western blotting, MALDI TOF-TOF, and circular dichroism spectroscopy analysis. Homology modeling and docking studies revealed that tagging GST with sFRP1 does not change the binding conformation of the cysteine-rich domain and hence, possibly does not alter its function. The novel anti-proliferative activity of GST-sFRP1 was demonstrated in a dose-dependent manner on two cancer cell lines, viz., HeLa (cervical cancer) and MCF-7 (breast cancer). Also, combination therapy of the protein with chemotherapeutic drugs resulted in enhanced anti-cancer activity. This opens up a new avenue in the application of recombinant sFRP1 for cancer therapeutics.

Purification and expression of glutathione S-transferase from a Malaysian population of Aedes albopictus (Diptera: Culicidae)

Glutathione S-transferase (GST) from the 4th instar larvae of the dengue vector Aedes albopictus was purified by glutathione-agarose affinity chromatography and characterised using SDS-PAGE. The expression of the purified enzyme in the life stages and insecticide treated populations of Ae. albopictus as well as its cross-reactivity with larval GST of two dipteran species Aedes aegypti and Batrocera papayae were observed using western blotting. The purified GST had a specific activity of 196.0 ± 11 μmol/min/mg with a purification fold and yield of 28 and 69%, respectively. The SDS-PAGE analysis of the purified GST depicted a single band size of 23 kDa. The GST was expressed in all the larval and adult stages of Ae. albopictus with the exception of the pupal stage. However, the expression level in the adult stage was visibly reduced as compared to the larval stages. Western blotting analysis showed no cross-reactivity with the GST of Ae. aegypti (4th instar) and B. papayae (3rd instar) larvae. The expression of this enzyme was not inducible by exposure to the insecticides dichlorodiphenyltrichloroethane (1.25 mg/L) and malathion (0.3125 mg/L).

Proteomic Profiling of Cytosolic Glutathione Transferases from Three Bivalve Species: Corbicula fluminea, Mytilus galloprovincialis and Anodonta cygnea

Suspension-feeding bivalves are considered efficient toxin vectors with a relative insensitivity to toxicants compared to other aquatic organisms. This fact highlights the potential role of detoxification enzymes, such as glutathione transferases (GSTs), in this bivalve resistance. Nevertheless, the GST system has not been extensively described in these organisms. In the present study, cytosolic GSTs isoforms (cGST) were surveyed in three bivalves with different habitats and life strategies: Corbicula fluminea, Anodonta cygnea and Mytilus galloprovincialis. GSTs were purified by glutathione-agarose affinity chromatography, and the collection of expressed cGST classes of each bivalve were identified using a proteomic approach. All the purified extracts were also characterized kinetically. Results reveal variations in cGST subunits collection (diversity and properties) between the three tested bivalves. Using proteomics, four pi-class and two sigma-class GST subunits were identified in M. galloprovincialis. C. fluminea also yielded four pi-class and one sigma-class GST subunits. For A. cygnea, two mu-class and one pi-class GST subunits were identified, these being the first record of GSTs from these freshwater mussels. The affinity purified extracts also show differences regarding enzymatic behavior among species. The variations found in cGST collection and kinetics might justify diverse selective advantages for each bivalve organism.

Molecular Cloning, Purification and Functional Implications of Recombinant GST Tagged hGMCSF Cytokine

We report the cloning, purification and cell proliferative activity of a novel recombinant GST tagged human granulocyte macrophage colony stimulating factor (GST-hGMCSF). The hGMCSF gene was PCR amplified from the cDNA of ACHN renal carcinoma cells and was cloned into the bacterial expression vector. The GST-hGMCSF was purified to homogeneity using glutathione agarose affinity chromatography and subsequently characterized by Western blot, circular dichroism (CD) and MALDI TOF-TOF analysis. Homology modelling studies revealed the possible binding domains of the recombinant cytokine with cognate receptor. The proliferation of THP-1, Raw 264.7, MCF-7 and U87MG cells upon GST-hGMCSF addition was found to be dose dependent. Hence, this functionally active recombinant cytokine has potential application in cancer therapy for stimulating facile growth recovery of normal cell population.

Hydrogel nanocarrier encapsulated recombinant IκBα as a novel anticancer protein therapeutics

The NFκB overexpression triggers drug resistance in many types of cancer by up-regulating anti-apoptotic genes. IκBα, an endogenous protein and natural inhibitor of NFκB, blocks translocation of NFκB from cytoplasm to nucleus and thereby, sensitizes cancer cells to external stimuli. Herein, we have cloned a PCR amplified cDNA of IκBα, and expressed the recombinant GST tagged IκBα in Escherichia coli BL21 (DE3) cells. The recombinant GST-IκBα was purified by glutathione-agarose affinity chromatography and characterized by Western blot, MALDI-TOF, UV-Vis spectroscopy and circular dichroism spectroscopy. The GST-IκBα recombinant protein was encapsulated within polyvinyl alcohol (PVA)/polyvinyl pyrrolidone (PVP) hydrogel nanocarrier (NC) and then characterized by transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS) and zeta potential measurements. The pH-dependent protein release was performed in vitro to study the pH tunability of the hydrogel NCs. Furthermore, the therapeutic efficacy of GST-IκBα loaded hydrogel NCs was evaluated on HeLa (cervical carcinoma) cells by cytotoxicity assay and cell cycle analysis. TUNEL assay by flow cytometry confirmed apoptosis of HeLa cells. Administration of GST-IκBα by hydrogel NCs showed significant cell growth inhibition of drug resistant U87MG cells in combination with 5-FU. Our results essentially attributed the hydrogel NC-mediated administration of recombinant GST-IκBα as a novel recombinant protein therapeutic approach for cancer, which may further be regimented in combination therapy.

Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study

Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and KM and Vmax values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC50 values, Ki constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag+; Cd2+, Cr2+ and Mg2+.

Purification and Biochemical Characterization of Glutathione S-Transferases inBactrocera minax(Diptera: Tephritidae)

Glutathione S-transferases (GSTs) were purified from 3 developmental stages of Bactrocera minax through glutathione-agarose affinity chromatography, and characterized subsequently using the Michaelis-Menten kinetics toward the artificial substrates 1-chloro-2, 4-dinitrobenzene (CDNB) and reduced glutathione (GSH), respectively. Compared to the counterparts of third instar larva and adult, the highest specific activity of the purified GSTs towards CDNB was observed in the pupae. Although the specific activities of purified enzymes varied among 3 developmental stages, the purification yields were similar. SDS-PAGE revealed only one band at 23 kDa for all 3 stages. GSTs of the adults exhibited the highest Km value towards CDNB, while for GSH the pupae possessed the highest Km. The optimum temperature and pH for CDNB conjugation of the 3 stages were 37 °C and 7.5, respectively. Inhibition kinetics showed that ethacrynic acid, bromosulfalein, diethyl maleate, tetraethylthiuram disulfide and curcumin possessed excellent inhibitory effects on purified GSTs in B. minax. Moreover, the pupa showed the highest catalytic capability based on Vmax values for both the CDNB and GSH, which may suggest a potentially higher GSTs detoxification ability in the pupal stage than the other 2 developmental stages.

The effect of some antineoplastic agents on glutathione S-transferase from human erythrocytes

Glutathione S-transferase was purified from human erythrocytes and effects of some antineoplastic agents were investigated on the enzyme activity. The purification procedure was composed of Glutathione-Agarose affinity chromatography after preparation of erythrocytes hemolysate. Using this procedure, the enzyme, having the specific activity of 16.00 EU/mg proteins, was purified 1143-fold with a yield of 80%. The purified enzyme showed a single band on the SDS-PAGE. The effects of paclitaxel, cyclophosphamide, and gemcitabine, are antineoplastic agents, were examined on the in vitro enzyme activity of glutathione S-transferase and were determined to be inhibitors for the enzyme. IC50 values were 0.23 mM for paclitaxel, 5.57 mm for cyclophosphamide, and 6.35 mM for gemcitabine. These constants were 0.182 ± 0.028 mM and 0.162 ± 0.062 mM for paclitaxel, 6.97 ± 0.49 mM and 10.50 ± 5.43 mM for cyclophosphamide, and 6.71 mM and 7.93 mM for gemcitabine, with GSH and CDNB substrates, respectively. Inhibition types of all inhibitors were noncompetitive.

Purification and biochemical characterization of glutathione S‐transferases from four field populations of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

Glutathione S-transferases (GSTs) are a group of detoxification enzymes that catalyze the nucleophilic addition of glutathione to a wide variety of endogenous and xenobiotic compounds. In this study, GSTs were purified from four field populations of Bactrocera dorsalis with different insecticide susceptibilities by glutathione-agarose affinity chromatography. The populations were collected from Dongguan (DG) and Guangzhou (GZ) of the Guangdong Province, Haikou of the Hainan province (HN), and Kunming of the Yunnan province (YN), China. Differences in GST characteristics among the four populations were studied using purified enzyme samples through comparative SDS-PAGE, kinetic, and inhibition experiments. The specific activities of the purified enzymes were similar, but the purification yield of the GZ population (31.54%) was the lowest. SDS-PAGE analysis showed only one band at approximately 23 kDa for these four populations. Kinetic analyses showed that the affinities of the purified GSTs from the GZ and YN populations for 1-chloro-2.4-dinitrobenzene (CDNB) were much higher than those of GSTs from the other two populations, whereas the HN population had the highest catalytic capability in terms of V(max) value. The optimum temperature for CDNB conjugation was 37 °C and the optimum pH was 7.5 in all four populations. Inhibition kinetics showed that ethacrynic acid, diethyl maleate, tetraethylthiuram disulfide, curcumin, bromosulfalein, and β-cypermethrin had excellent inhibitory effects on GSTs in the four populations of B. dorsalis, but the low inhibitory effects of malathion and avermectin did not differ between populations. These results suggest that GSTs may have a role in detoxification of β-cypermethrin in B. dorsalis.