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1.
Abstract: An enzyme that possesses glutathione S -transferase (GST) activity was found in the fall webworm, Hyphantria cunea . The enzyme was purified to homogeneity for the first time by ammonium sulphate fractionation and affinity chromatography. The N-terminal sequence of the purified protein was similar to those of Sigma-class GSTs. The purified GST retained more than 75% of its original GST activity after incubation at pH 5–8. Incubation for 30 min at temperatures below 50°C scarcely affected the activity. The enzyme was able to catalyse the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. 相似文献
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The tyrosine kinase activity of the epidermal-growth-factor receptor is necessary for phospholipase A2 activation.
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H J Goldberg M M Viegas B L Margolis J Schlessinger K L Skorecki 《The Biochemical journal》1990,267(2):461-465
Cytosolic glutathione transferases (GSTs) were purified from the rat spleen by S-hexyl-GSH-Sepharose chromatography, and two major forms were identified as GSTs 2-2 and 7-7 (GST P). Besides these forms an acidic form (pI 5.8) was purified by chromatofocusing at pH 7-4 and it accounted for about 1% of the total GST activity bound to S-hexyl-GSH-Sepharose. Two-dimensional gel electrophoresis revealed that it is a homodimer (subunit Mr 26,000 with pI 5.8). Immunoblot analysis demonstrated that it was immunologically related to GSTs 2-2 and 1-1, and its N-terminal amino acid was apparently blocked, similarly to other forms of the class Alpha. This form had a low activity towards cumene hydroperoxide or 4-hydroxynon-2-enal, indicating that this form differed from GSTs 10-10 and 8-8 as well as from GSTs 1-1 and 2-2. These results suggest that it is a new form of GST belonging to the class Alpha. 相似文献
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T Igarashi A Kohara Y Shikata F Sagami J Sonoda T Horie T Satoh 《The Journal of biological chemistry》1991,266(32):21709-21717
In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes. 相似文献
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Tripti Pandey Sudhir Kumar Singh Gaurav Chhetri Timir Tripathi Arvind Kumar Singh 《PloS one》2015,10(5)
Glutathione S-transferases (GSTs) are multifunctional enzymes present in virtually all organisms. Besides having an essential role in cellular detoxification, they also perform various other functions, including responses in stress conditions and signaling. GSTs are highly studied in plants and animals; however, the knowledge regarding GSTs in cyanobacteria seems rudimentary. In this study, we report the characterization of a highly pH stable GST from the model cyanobacterium- Synechocystis PCC 6803. The gene sll0067 was expressed in Escherichia coli (E. coli), and the protein was purified to homogeneity. The expressed protein exists as a homo-dimer, which is composed of about 20 kDa subunit. The results of the steady-state enzyme kinetics displayed protein’s glutathione conjugation activity towards its class specific substrate- isothiocyanate, having the maximal activity with phenethyl isothiocyanate. Contrary to the poor catalytic activity and low specificity towards standard GST substrates such as 1-chloro-2,4-dinitrobenzene by bacterial GSTs, PmGST B1-1 from Proteus mirabilis, and E. coli GST, sll0067 has broad substrate degradation capability like most of the mammalian GST. Moreover, we have shown that cyanobacterial GST sll0067 is catalytically efficient compared to the best mammalian enzymes. The structural stability of GST was studied as a function of pH. The fluorescence and CD spectroscopy in combination with size exclusion chromatography showed a highly stable nature of the protein over a broad pH range from 2.0 to 11.0. To the best of our knowledge, this is the first GST with such a wide range of pH related structural stability. Furthermore, the presence of conserved Proline-53, structural motifs such as N-capping box and hydrophobic staple further aid in the stability and proper folding of cyanobacterial GST- sll0067. 相似文献
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E. V. Pradedova O. D. Nimaeva I. S. Truchan R. K. Salyaev 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2016,10(3):223-232
Glutathione transferase (GST) activity revealed in vacuoles of red beetroot (Beta vulgaris L.) cells was investigated in comparison with the GST activity of plastids and extracts of tissues. The level of GST activity determined by spectrophotometric method proved fairly high in water extracts and membrane fractions of isolated vacuoles and plastids, as well as in water extracts of tissues. In the objects studied, pH dependence of the GST activity slightly differed. Optimal pH for the vacuolar GST activity was in the range 7.0–7.5, for the GST of plastids and tissue extracts it was 7.5. The GSTs differed in specificity to the substrates fluorodifen and ethacrynic acid. The activity of the vacuolar and tissue extract GSTs with fluorodifen was significantly higher than that of the GST from plastids. Ethacrynic acid, often used as a competitive inhibitor of GST, almost completely inhibited the GST activity assayed with 1-chloro-2,4-dinitrobenzene as a main substrate. However, ethacrynic acid was a substrate only for the GSTs of vacuoles and tissue extract, but not for the GST of plastids. Using zymography allowing estimation of the GST activity in a gel after electrophoresis of proteins, several zones of enzymatic activity were revealed in all objects that may correspond to different isozymes. It was found that the composition of the vacuolar GST isoforms and their substrate specificity may differ from the GSTs of other cellular structures. It is assumed that vacuole, having quite high activity of GST, should make a significant contribution to intracellular detoxification processes. 相似文献
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K Tamai K Satoh S Tsuchida I Hatayama T Maki K Sato 《Biochemical and biophysical research communications》1990,167(1):331-338
Treatment of Class Pi glutathione S-transferases (GST) such as rat GST P (7-7), human GST pi and mouse GST MII with 0.05-0.1 mM N-ethylmaleimide (NEM) in 0.1 M Tris-HCl (pH 7.8) resulted in almost complete inactivation of these forms, whereas no or less inactivation occurred for GSTs in Class Alpha and Mu under the same conditions. Inactivated GST P lost its S-hexyl-GSH-Sepharose column affinity. About 0.8 mol of [14C]NEM was found to be covalently bound to 1 mol of GST P subunit when 80% of the activity was lost. Similar treatment with N-dimethyl-amino-3,5-dinitrophenyl maleimide, a colored analogue of NEM, followed by trypsin digestion, HPLC and amino acid sequence analysis revealed that one cysteine residue at the 47th position from the N-terminal of the GST P subunit was preferentially modified. Subunits of GST P and GST pi are known to have 4 cysteine residues at the same corresponding positions. The present results suggest that the 47th cysteine residue may be located in the vicinity of the active site of Class Pi GSTs. 相似文献
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GSTs from adult Drosophila melanogaster have been partially purified using three different affinity chromatography media and separated by 2-DE. Nine GSTs have been identified by MALDI-TOF MS. In the absence of special treatments, eight GSTs could be positively identified. These were DmGSTs D1 (the dominant Delta isoform which was present in five protein zones of differing pI) and D3 (and possibly also D5); the Epsilon-class GSTs E3, 6, 7 and 9 and a previously uncharacterised, probable member of the class, CG16936. The Sigma-class DmGSTS1 was prominent. DmGSTD2 was detected only after pretreatment of the flies with Phenobarbital (PhB). Treatment with Paraquat (PQ) led to an increase in the total GST activity, as measured with the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloro-nitrobenzene (DCNB) and an increase in the relative amounts of the D1, D3, E6 and E7 isoforms. PhB treatment led to increases in the relative amounts of the D1, D2, E3, E6, E7 and E9 isoforms detected with a possible depression in the relative amount of GSTS1. CG16936 was unaffected by either pretreatment. 相似文献
11.
B P Bowman T W Snell B J Cochrane 《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,95(3):619-624
1. The enzyme glutathione S-transferase (GST), a critical element in xenobiotic metabolism, was isolated from the marine rotifer Brachionus plicatilis and its freshwater congener B. calyciflorus. 2. In B. plicatilis, GST comprised 4.2% of cytosolic protein and was present as three separate isozymes with mol. wts 30,000, 31,400 and 33,700. Specific activity of crude homogenates was 56 nmol min-1 mg-1 protein, while that of affinity chromatography purified GST was 1850. 3. In B. calyciflorus, GST was present as two isozymes with mol. wts of 26,300 and 28,500, representing 1.0% of cytosolic protein. Crude GST specific activity was 1750 nmol min-1 mg-1 protein and purified was 72,400. 4. Rotifer GSTs are unusual because they are monomers whereas all other animals thus far investigated posses dimeric GSTs. 相似文献
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Glutathione transferases (GSTs) catalyze the bioactivation of the thiopurine prodrugs azathioprine, cis-6-(2-acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG), thereby releasing the antimetabolites 6-mercaptopurine and 6-thioguanine. In the GST Mu class, GST M1-1 has the highest catalytic efficiency, whereas GST M2-2 and other enzymes are less active. In the evolution of Mu class GSTs, residue 210 appears hypervariable and has particular functional significance. We demonstrate that the catalytic activity of GST M1-1 with cAVTP or tAVTG is successively diminished when wild-type Ser-210 is mutated into Ala followed by Thr. Conversely, mutating wild-type Thr-210 in GST M2-2 into Ala and Ser enhanced the corresponding activities. Comparisons were also made with GST M2-2 distinguished by Gly or Pro in position 210, as well as wild-type GSTs M4-4 and M5-5. The results suggest that the hydroxyl group of Ser in position 210 stabilizes the transition state of the GST-catalyzed reaction. The low activity of GSTs containing Thr in position 210 is probably due to steric hindrance caused by the beta-methyl group of the side chain. The ratios of the different catalytic efficiencies were translated into differences in the Gibbs free energies of transition state stabilization. The effects of the mutations were qualitatively parallel for the alternative substrates, but vary significantly in magnitude. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs. 相似文献
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Paragonimus westermani: a cytosolic glutathione S-transferase of a sigma-class in adult stage 总被引:2,自引:0,他引:2
Hong SJ Kang SY Chung YB Chung MH Oh YJ Kang I Bahk YY Kong Y Cho SY 《Experimental parasitology》2000,94(3):180-189
We purified cytosolic glutathione S-transferase (GST) of adult Paragonimus westermani monitoring its activity with 1-chloro-2,4-dinitrobenzene (CDNB). The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21% recovery rate through a three-step procedure. The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6. Monoclonal antibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from other helminths. cDNA library was constructed in lambdaZAP II bacteriophage and screened with anti-Pw28GST. The corresponding gene containing a single open reading frame of 804 bp encoded 211 amino acids. The predicted amino acid sequence exhibited a higher homology with catalytic domain near N-terminus of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%). The sequence contained both Tyr-6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs. The apparent K(m) value of a recombinant enzyme was 0.78 mM. Both native and recombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity against epoxy-3-(p-nitrophenoxy)-propane. The activities were inhibited by bromosulfophthalein, cibacron blue, and albendazole, but not by praziquantel. These findings indicate that adult P. westermani has a class sigma GST. 相似文献
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Characterisation of a zeta class glutathione transferase from Arabidopsis thaliana with a putative role in tyrosine catabolism 总被引:7,自引:0,他引:7
A glutathione transferase (GST) similar to zeta GSTs in animals and fungi has been cloned from Arabidopsis thaliana using RT-PCR. The Arabidopsis zeta GST (AtGSTZ1) was expressed in Escherichia coli as his-tagged polypeptides, which associated together to form the 50-kDa AtGSTZ1-1 homodimer. Following purification, AtGSTZ1-1 was assayed for a range of activities and compared with other purified recombinant plant GSTs from the phi, tau, and theta classes. AtGSTZ1-1 differed from the other GSTs in showing no glutathione conjugating activity toward xenobiotics and no glutathione peroxidase activity toward organic hydroperoxides. Uniquely among the plant GSTs, AtGSTZ1-1 showed activity as a maleylacetone isomerase (MAI). This glutathione-dependent reaction is analogous to the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate, which occurs in the course of tyrosine catabolism to acetoacetate and fumarate. Thus, rather than functioning as a conventional GST, AtGSTZ1-1 appears to be involved in tyrosine degradation. In addition to the MAI activity, the AtGSTZ1-1 also catalyzed the glutathione-dependent dehalogenation of dichloroacetic acid to glyoxylic acid. This latter activity was used to demonstrate the presence of functional AtGSTZ1-1 inplanta. 相似文献
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Ajamaluddin Malik Haseeb Jagirdar Nayyar Rabbani Mohd Shahnawaz Khan Anwar Ahmed Abdulrahman M. Al-Senaidy 《Preparative biochemistry & biotechnology》2013,43(7):650-666
Glutathione S-transferases (GSTs) are multifunctional enzymes and play an important role in cellular detoxification. Besides this, GSTs act as cytosolic carrier proteins that bind hydrophobic compounds such as heme, bilirubin, steroids, and polycyclic hydrocarbons. GST has great importance in biotechnology, as it is a target for vaccine and drug development and biosensors development for xenobiotics. Moreover, the GST tag has been extensively used for protein expression and purification. Until now, biophysical properties of camel liver GST have not been characterized. In the present study we have purified camel (Camelus dromedarius) liver GST to homogeneity in a single step by affinity chromatography with 23.4-fold purification and 60.6% yield. Our results showed that maximal activity of GST was at pH 6.5 and it was stable in the pH range of 5 to 10. The optimum temperature was 55°C and the Tm was 57°C. The chemical chaperone glycerol (3.3 M) was able to protect GST activity and aggregation against thermal denaturation by stabilizing the protein structure at 50 and 57°C, respectively. However, L-arginine (125 mM) did not protect GST against thermal stress. Far-ultraviolet circular dichroism (CD) spectra showed that glycerol protected the secondary structure of GST while L-arginine induced conformational changes under thermal stress. In conclusion, our studies on the GST stability suggest that glycerol works as a stabilizer and L-arginine acts as a destabilizer. 相似文献
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A new Anopheles dirus glutathione S-transferase (GST) has been obtained and named adGST4-1. Both genomic DNA and cDNA for heterologous expression were acquired. The genomic sequence was 3188bp and consisted of the GST gene as well as flanking sequence. The flanking sequence was analyzed for possible regulatory elements that would control gene expression. In Drosophila several of these elements have been shown to be involved in development and cell differentiation. The deduced amino acid sequence has low identity compared with the four alternatively spliced enzymes, adGST1-1 to 1-4, from another An. dirus GST gene adgst1AS1. The percent identities are 30--40% and 11--12% comparing adGST4-1 to insect GSTs from Delta and Sigma classes, respectively. Enzyme characterization of adGST4-1 shows it to be distinct from the other An. dirus GSTs because of low enzyme activity for customary GST substrates including 1-chloro-2, 4-dinitrobenzene (CDNB). However, this enzyme has a greater affinity of interaction with pyrethroids compared to the other An. dirus GSTs. 相似文献
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Arna Runarsdottir 《Journal of molecular biology》2010,401(3):451-464
Glutathione transferases (GSTs) are known as promiscuous enzymes capable of catalyzing the conjugation of glutathione with a broad range of electrophilic substrates. A previous study based on recombinant chimeras derived from human GST M1-1 and GST M2-2 demonstrated the formation of a subset of F1 generation GSTs, which had lost high activity with substrates distinguishing parental enzymes. In the present study, the members of this subset were recombined by DNA shuffling to produce an F2 generation of GSTs. Screening of 930 bacterial clones demonstrated that 83% of recombinant enzyme variants were active with at least one of three alternative substrates: phenethyl isothiocyanate (PEITC), 1-chloro-2,4-dinitrobenzene, or p-nitrophenyl acetate. The majority had similar low activity as the parental GSTs in the F1 generation. However, 17 novel enzymes displayed high activity with PEITC. Half of these enzymes were similar to GST M1-1, which also has high activity with the same substrate, and all of these GSTs featured Tyr116/Ser210 in the active site. This group of F2 variants apparently had reverted to the GST M1-1 type. A second group of F2 variants with high PEITC activity was characterized by His116 in the active site. This category represented a new variety of GSTs, which demonstrated higher selectivity for isothiocyanate substrates than the GST M1-1 type. The different groups of GSTs can be considered as distinct molecular quasi-species, each of which comprises variant amino acid sequences. The quasi-species are structurally distinguished by active-site residues that govern their substrate selectivities. Clearly, minimal alterations of the active site can generate enzymes with highly distinctive functional properties. 相似文献
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Redesign of glutathione transferases (GSTs) has led to enzymes with remarkably enhanced catalytic properties. Exchange of substrate-binding residues in GST A1-1 created a GST A4-4 mimic, called GIMFhelix, with >300-fold improved activity with nonenal and suppressed activity with other substrates. In the present investigation GIMFhelix was compared with the naturally-evolved GSTs A1-1 and A4-4 by determining catalytic efficiencies with nine alternative substrates. The enzymes can be represented by vectors in multidimensional substrate-activity space, and the vectors of GIMFhelix and GST A1-1, expressed in kcat/Km values for the alternative substrates, are essentially orthogonal. By contrast, the vectors of GIMFhelix and GST A4-4 have approximately similar lengths and directions. The broad substrate acceptance of GST A1-1 contrasts with the high selectivity of GST A4-4 and GIMFhelix for alkenal substrates. Multivariate analysis demonstrated that among the diverse substrates used, nonenal, cumene hydroperoxide, and androstenedione are major determinants in the portrayal of the three enzyme variants. These GST substrates represent diverse chemistries of naturally occurring substrates undergoing Michael addition, hydroperoxide reduction, and steroid double-bond isomerization, respectively. In terms of function, GIMFhelix is a novel enzyme compared to its progenitor GST A1-1 in spite of 94% amino-acid sequence identity between the enzymes. The redesign of GST A1-1 into GIMFhelix therefore serves as an illustration of divergent evolution leading to novel enzymes by minor structural modifications in the active site. Notwithstanding low sequence identity (60%), GIMFhelix is functionally an isoenzyme of GST A4-4. 相似文献
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Molecular cloning and characterization of three sigma glutathione S-transferases from disk abalone (Haliotis discus discus) 总被引:1,自引:0,他引:1
Wan Q Whang I Lee J 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2008,151(3):257-267
Three novel glutathione S-transferase (GSTs) cDNAs were cloned from a disk abalone (Haliotis dicus discus) cDNA library. Multiple alignment and phylogenetic analysis of three GSTs revealed that their closest relationship is with insect sigma GSTs. Recombinant GSTs were over-expressed in Escherichia coli as soluble fusion proteins. HdGSTS1 and HdGSTS2 were active towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid, whereas HdGSTS3 appeared to be a non-enzymatic GST. Two active GSTs had similar optimum conditions for enzymatic reaction at pH 8.0 and temperature of approximately 30 degrees C. Molecular modeling analysis of three GSTs implicates their diverse active sites as being responsible for their different enzymatic features. Three sigma GSTs had significantly different expression patterns and levels of expression in abalone tissues, indicating their different functions. After 48 h-exposure to three model marine pollutants, only HdGSTS1 exhibited a proper inducibility, exhibiting its good biomarker potential for organic contaminants in marine environment. In contrast, the other two sigma GSTs revealed a minor role in the response of pollutants exposure. 相似文献