Mammalian GSTs Research Articles

Structure and steroid isomerase activity of Drosophila glutathione transferase E14 essential for ecdysteroid biosynthesis.

Ecdysteroids are critically important for the formation of the insect exoskeleton. Cholesterol is a precursor of ecdysone and its active form 20-hydroxyecdysone, but some steps in the ecdysteroid biosynthesis pathway remain unknown. An essential requirement of glutathione (GSH) transferase GSTE14 in ecdysteroid biosynthesis has been established in Drosophilamelanogaster, but its function is entirely unknown. Here, we have determined the crystal structure of GSTE14 in complex with GSH and investigated the kinetic properties of GSTE14 with alternative substrates. GSTE14 has high-ranking steroid double-bond isomerase activity, albeit 50-fold lower than the most efficient mammalian GSTs. Corresponding steroid isomerizations are unknown in insects, and their exact physiological role remains to be shown. Nonetheless, the essential enzyme GSTE14 is here demonstrated to be catalytically competent and have a steroid-binding site.

Pharmacophore-Based Virtual Screening of Novel GSTP1-1 Inhibitors

Glutathione transferase enzymes play an important role in metabolism and detoxification of numerous xenobiotics, electrophilic drugs, environmental carcinogens, and products of oxidative stress in living organisms. Human GST P1-1 is the most prevalent isoform of the mammalian cytosolic GSTs and this enzyme participates in a particular role in one of the mechanisms of the development of resistance in cancer cells toward the administration of anticancer agents in chemotherapy. Herein, pharmacophore analysis were performed using bioactive conformation of the known inhibitor of GSTP1-1, ethacrynic acid (pdb ID:2GSS). Phase module of the Schrödinger suite was used to generate pharmacophore hypothesis. Molecules with same pharmacophoric features were screened from among the commercially available compounds in the ZINC database and ligand filtration was also done to obtain an efficient collection of hit molecules by employing Lipinski “rule of five” using Qikprop module. As a result, some of the compounds obtained from this study, could be the promising inhibitors of hGSTP1-1 enzyme.

Two glutathione transferase isoforms isolated from juvenile cysts of Taenia crassiceps: identification, purification and characterization.

We identified and characterized the first two glutathione transferases (GSTs) isolated from juvenile cysts of Taenia crassiceps (EC 2.5.1.18). The two glutathione transferases (TcGST1 and TcGST2) were purified in a single-step protocol using glutathione (GSH)-sepharose chromatography in combination with a GSH gradient. The specific activities of TcGST1 and TcGST2 were 26 U mg-1 and 19 U mg-1, respectively, both at 25°C and pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates. The Km(CDNB) and Kcat(CDNB) values for TcGST1 and TcGST2 (0.86 μm and 62 s-1; 1.03 μm and 1.97 s-1, respectively) and Km(GSH) and Kcat(GSH) values for TcGST1 and TcGST2 (0.55 μm and 11.61 s-1; 0.3 μm and 32.3 s-1, respectively) were similar to those reported for mammalian and helminth GSTs. Mass spectrometry analysis showed that eight peptides from each of the two parasite transferases were a match for gi|29825896 glutathione transferase (Taenia solium), confirming that both enzymes are GSTs. The relative molecular masses were 54,000 ± 0.9 for the native enzymes and 27,500 ± 0.5 for the enzyme subunits. Thus, TcGST1 and TcGST2 are dimeric proteins. Optimal TcGST1 and TcGST2 activities were observed at pH 8.5 in the range of 20-55°C and pH 7.5 at 35-40°C, respectively. TcGST1 and TcGST2 were inhibited by cibacron blue (CB), bromosulphophthalein (BST), rose bengal (RB), indomethacin and haematin (Hm) with 50% inhibitory concentrations (IC50) in the μm range. TcGST1 was inhibited in a non-competitive manner by all tested inhibitors with the exception of indomethacin, which was uncompetitive. The discovery of these new GSTs facilitates the potential use of T. crassiceps as a model to investigate multifunctional GSTs.

Characterization of porcine Alpha-class glutathione transferase A1-1

An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissues and animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence of our clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to our cloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1∗. The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactions was at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.

Mammalian Cytosolic Glutathione Transferases

Glutathione Transferases (GSTs) are crucial enzymes in the cell detoxification process catalyzing the nucleophilic attack of glutathione (GSH) on toxic electrophilic substrates and producing a less dangerous compound. GSTs studies are of great importance since they have been implicated in the development of drug resistance in tumoral cells and are related to human diseases such as Parkinson's, Alzheimer's, atherosclerois, liver cirrhosis, aging and cataract formation. In this review we start by providing an evolutionary perspective of the mammalian cytosolic GSTs known to date. Later on we focus on the more abundant classes alpha, mu and pi and their structure, catalysis, metabolic associated functions, drug resistance relation and inhibition methods. Finally, we introduce the recent insights on the GST class zeta from a metabolic perspective.

Binding and Glutathione Conjugation of Porphyrinogens by Plant Glutathione Transferases

Overexpression in Escherichia coli of a tau (U) class glutathione transferase (GST) from maize (Zea mays L.), termed ZmGSTU1, caused a reduction in heme levels and an accumulation of porphyrin precursors. This disruption was highly specific, with the expression of the closely related ZmGSTU2 or other maize GSTs having little effect. Expression in E. coli of a series of chimeric ZmGSTU1/ZmGSTU2 proteins identified domains responsible for disrupting porphyrin metabolism. In addition to known heme precursors, expression of ZmGSTU1 led to the accumulation of a novel glutathione conjugate of harderoporphyrin(ogen) (2,7,12,18-tetramethyl-3-vinylporphyrin-8,13,17-tripropionic acid). Using the related protoporphyrinogen as a substrate, conjugation could be shown to occur on one vinyl group and was actively catalyzed by the ZmGSTU. In plant transgenesis studies, the ZmGSTUs did not perturb porphyrin metabolism when expressed in the cytosol of Arabidopsis or tobacco. However, expression of a ZmGSTU1-ZmGSTU2 chimera in the chloroplasts of tobacco resulted in the accumulation of the harderoporphyrin(ogen)-glutathione conjugate observed in the expression studies in bacteria. Our results show that the well known ability of GSTs to act as ligand binding (ligandin) proteins of porphyrins in vitro results in highly specific interactions with porphyrinogen intermediates, which can be demonstrated in both plants and bacteria in vivo.

Transition State Model and Mechanism of Nucleophilic Aromatic Substitution Reactions Catalyzed by Human Glutathione S-Transferase M1a-1a

An active site His107 residue distinguishes human glutathione S-transferase hGSTM1-1 from other mammalian Mu-class GSTs. The crystal structure of hGSTM1a-1a with bound glutathione (GSH) was solved to 1.9 A resolution, and site-directed mutagenesis supports the conclusion that a proton transfer occurs in which bound water at the catalytic site acts as a primary proton acceptor from the GSH thiol group to transfer the proton to His107. The structure of the second substrate-binding site (H-site) was determined from hGSTM1a-1a complexed with 1-glutathionyl-2,4-dinitrobenzene (GS-DNB) formed by a reaction in the crystal between GSH and 1-chloro-2,4-dinitrobenzene (CDNB). In that structure, the GSH-binding site (G-site) is occupied by the GSH moiety of the product in the same configuration as that of the enzyme-GSH complex, and the dinitrobenzene ring is anchored between the side chains of Tyr6, Leu12, His107, Met108, and Tyr115. This orientation suggested a distinct transition state that was substantiated from the structure of hGSTM1a-1a complexed with transition state analogue 1-S-(glutathionyl)-2,4,6-trinitrocyclohexadienate (Meisenheimer complex). Kinetic data for GSTM1a-1a indicate that kcat(CDNB) for the reaction is more than 3 times greater than kcat(FDNB), even though the nonenzymatic second-order rate constant is more than 50-fold greater for 1-fluoro-2,4-dinitrobenzene (FDNB), and the product is the same for both substrates. In addition, Km(FDNB) is about 20 times less than Km(CDNB). The results are consistent with a mechanism in which the formation of the transition state is rate-limiting in the nucleophilic aromatic substitution reactions. Data obtained with active-site mutants support transition states in which Tyr115, Tyr6, and His107 side chains are involved in the stabilization of the Meisenheimer complex via interactions with the ortho nitro group of CDNB or FDNB and provide insight into the means by which GSTs adapt to accommodate different substrates.

Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class.

From the fruitfly, Drosophila melanogaster, ten members of the cluster of Delta-class glutathione S-transferases (GSTs; formerly denoted as Class I GSTs) and one member of the Epsilon-class cluster (formerly GST-3) have been cloned, expressed in Escherichia coli, and their catalytic properties have been determined. In addition, nine more members of the Epsilon cluster have been identified through bioinformatic analysis but not further characterized. Of the 11 expressed enzymes, seven accepted the lipid peroxidation product 4-hydroxynonenal as substrate, and nine were active in glutathione conjugation of 1-chloro-2,4-dinitrobenzene. Since the enzymically active proteins included the gene products of DmGSTD3 and DmGSTD7 which were previously deemed to be pseudogenes, we investigated them further and determined that both genes are transcribed in Drosophila. Thus our present results indicate that DmGSTD3 and DmGSTD7 are probably functional genes. The existence and multiplicity of insect GSTs capable of conjugating 4-hydroxynonenal, in some cases with catalytic efficiencies approaching those of mammalian GSTs highly specialized for this function, indicates that metabolism of products of lipid peroxidation is a highly conserved biochemical pathway with probable detoxification as well as regulatory functions.

Amino acid sequence of the major form of toad liver glutathione transferase

To investigate structural relationship between amphibian and mammalian GSTs the complete amino acid sequence of the major form of glutathione transferase present in toad liver ( Bufo bufo) was determined. The enzyme subunit is composed of 210 amino acid residues corresponding to a molecular mass of 24,178 Da. In comparison with the primary structure of amphibian bbGSTP1-1, toad liver GST showed 54% sequence identity. On the other hand, toad liver GST showed about 45–55% sequence identity when compared with other pi class GST and less then 25% identity with GST of other classes. Amino acid residues involved in the H site and in the key and lock structure of the toad enzyme are significantly different from those of bbGSTP1-1 and other mammalian pi class GST. On the basis of its structural and immunological properties the toad liver GST, indicated as bbGSTP2-2, could represent the prototype of a subset of the pi family.

Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis.

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359 Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

Characterization of a recombinant mu-class glutathione S-transferase from Taenia solium.

A Taenia solium larval glutathione S-transferase fraction (SGSTF), composed of two proteins with Mr 25,500 (SGSTM1) and 26,500 (SGSTM2), was purified by GSH-sepharose. Its N-terminal sequence analysis revealed that both proteins are related to mammalian mu-class GST enzymes. A cDNA clone coding for SGSTM1 was isolated and the amino acid sequence analysis showed close identity with two Echinococcus GSTs and also high identity with several mu-class GSTs that have been reported. In addition, SGSTM1 presents a similar structure to mu-class GSTs, including the mu loop. The recombinant SGSTM1 is a dimeric protein with enzymatic properties clearly related to mammalian mu-class GSTs. Western blot studies indicated that SGSTM1 is not antigenically related to SGSTM2 or mammalian GSTs from rabbit, pig and rat livers. Immunization with SGSTF and SGSTM2 was highly effective in reducing cysticerci load in murine cysticercosis. In contrast, no protection was obtained using native SGSTM1 and recombinant SGSTM1 as immunogens.

A novel glutathione transferase from Haemophilus influenzae which has high affinity towards antibiotics

Cytosolic glutathione transferase (GSTs) are a family of multi-functional proteins which catalyse the conjugation of glutathione (GSH) to a large variety of endogenous and exogenous electrophilic compounds. Much is known about cytosolic mammalian GSTs, however, the presence of GSTs in several aerobic and anaerobic micro-organisms has also been demonstrated. Several findings seem to suggest that bacterial GSTs are involved in processes of biodegradation of xenobiotics, including antibiotics. However, the function played by these enzymes in the bacterial cell still remains to be clarified. At present, it is ill-defined whether bacterial GST can be classified, as in the case of mammalian enzymes, into several distinct classes. Here we report the purification of a GST isoform from Haemophilus influenzae using GSH-affinity chromatography. The purified protein was characterised by immunological and kinetic properties different from other known GSTs. The dissociation constants of chloramphenicol, ampicillin, rifampicin and tetracycline to the purified enzyme were 0.62, 9.06, 4.08 and 1.77 μM, respectively, as determined by following the quenching of the protein intrinsic fluorescence. These values were much lower than those previously determined for the same drugs with other mammalian or bacterial GSTs. The present results indicate that the enzyme purified from H. influenzae is a novel GST isoform well distinguished from other known mammalian or bacterial GSTs.

In Vitro Inhibition of Mammalian Glutathione Transferases by Selected Nitrobenzenes

Five structurally related nitrobenzenes (1,2-dinitrobenzene, 1,3-dinitrobenzene, 1,4-dinitrobenzene, 1,3,5-trinitrobenzene, and picric acid) and Meisenheimer complex [1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate] were evaluated as possible inhibitors of affinity purified mammalian glutathione transferases (GSTs) isolated from human liver or human term placenta and rat fiver. The results suggest that the degree of GST inhibition depends upon both the chemical in question and the enzyme source. Among the nitrobenzenes tested, 1,3,5-trinitrobenzene (TNB) was found to be the most potent inhibitor of GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) from all the sources, whereas 1,3-dinitrobenzene (1,3-DNB) was the least effective. TNB-caused inhibition of GST activity toward CDNB appeared to be isozyme specific in that compared to the enzyme from human term placenta (GSTP1–1), the degree of inhibition of the mixture of GST isozymes present in the fivers of adult rats and humans was low. The enzyme assays conducted with 3,4-dichloro-1-nitrobenzene (DCNB), ethacrynic acid (EA), and 4-nitropyridinei N-oxide also suggested the isozymespecific inhibition of rat fiver GST activity by TNB. The nature of TNB-caused inhibition of GSTP1–1 was competitive with respect to CDNB and yielded a Ki value of 12.5 θ M. With EA, a specific substrate for GSTP1–1, an IC50 value of ∼ 16 θ M was estimated for the GSTP1–1 inhibition by TNB. The Meisenheimer complex, the product of nonenzymatic GSH conjugation with TNB by different GSTs, was found to be the most potent inhibitor of mammalian GSTs, and IC50 values ranged between 1 and 4 θ M when the enzyme activity was assayedusing CDNB. The nature of GSTP1–1 inhibition was noncompetitive with respect to CDNB, with a Ki value of 1 θ M for Meisenheimer complex. Although a precise mechanism was not identified, it is postulated that GSH depletion and/or GST inhibition may contribute, at least partly, to the target organ toxicity caused by exposures of animals to different nitrobenzenes reported in the literature.

A piscine glutathione S-transferase which efficiently conjugates the end-products of lipid peroxidation

A cDNA clone for glutathione S-transferaseA (GSTA) from plaice ( Pleuronectes platessa) was expressed in Eschericia coli (E. coli) and purified to homogeneity by S-hexylglutathione affinity chromatography. When compared to literature values for a variety of purified mammalian GSTs, the heterologously expressed purified plaice enzyme had moderate activity towards the model substrate 1,2-chloro-2,4-dinitrobenzene (CDNB) and exhibited a Km of 2.5 ± 2 mM and Vmax of 30.9 ± 2.3 μmol min −1 mg −1. It had little or no activity towards several other model GST substrates including 1,2-dinitrochloro-4-benzene (DCNB), ethacrynic acid (EA), and p-nitrobenzylchloride (NBC). However plaice GSTA was a relatively efficient catalyst for the conjugation of a series of alk-2-enals and alk-2,4-dienals and also 4-hydroxynonenal. The highest activity observed with this series of substrates was with trans-non-2-enal with a Km of 17.9 ± 2.2 μM and a Vmax of 3.01 ± 0.57 μmol min −1 mg −1. These unsaturated alkenals have been identified in cells and cell extracts as highly toxic products arising from peroxidation of unsaturated fatty acids particularly during periods of oxidative stress. Fish are relatively rich in polyunsaturated fatty acids and thus GSTA mediated conjugation may be an important mechanism for detoxifying peroxidised lipid breakdown products.

THE ROLE OF ALPHA-CLASS GLUTATHIONE S-TRANSFERASES AGAINST OXIDATIVE STRESS

Cholesterol 7α- and 7β-hydroperoxides are mutagenic and cytotoxic steroids existing as minor components in rat skin and regarded as good aging markers in the rat. Rat liver and skin cytosols had high and very low activities of the reduction of cholesterol 7-hydroperoxides to the corresponding steroidal alcohols, respectively, in the presence of GSH. The GSH-dependent reduction of the steroid hydroperoxides in rat liver was catalyzed mainly by GSTs with the subunit A1(2) of the Alpha class, but not catalyzed by any other isoform of GST existing in rat liver. Se-containing GSH peroxidase (Se-GSH Px) also played a partial role, to a lesser extent than rat (r) GSTA1-3, in the GSH-dependent reduction of the hydroperoxides in rat liver cytosol. However, rat skin cytosol contained no detectable level of rGSTsAI-1(2) and Al-3, but contained a very low level of Se-GSH Px. The above facts strongly suggest that the age-dependent linear increase in the dermal levels of the cholesterol hydroperoxides as the aging markers in the rat is mainly responsible for the absence of rGSTs bearing the subunit A1(2) and for the very low level of Se-GSH Px in the skin. gpGSTA1-1 existed at a concentration of 1% of cytosolic protein in the liver of guinea pigs, which are well known to lack Se-GSH Px in their tissues. A comparative study using rGSTA1-2 and Theta-class rGSTT2-2 indicated gpGSTA1-1 to have the highest GSH Px activity toward cumene hydroperoxide of all known mammalian GSTs and also to have Px activity toward polyunsaturated fatty acid hydroperoxides (PUFAOOHs) comparable to rGSTT2-2, with the highest activity toward PUFA-OOHs of rGSTs. No detectable level of the GSTA4-4 ortholog highly active toward 4-hydroxy-2(E)-nonenal (4-HNE) was found in the liver of guinea pigs. All gpGST isoforms isolated from hepatic cytosol showed modest GSH-conjugating activity toward 4-HNE.

Purification and Characterisation of Hepatic Glutathione Transferases from a Herbivorous Marsupial, the Brushtail Possum ( Trichosurus vulpecula)

A single glutathione transferase isoenzyme was purified from hepatic cytosol of the brushtail possum and shown to represent 3.6 ± 0.3% of the total cytosolic protein. Characterisation of the enzyme, termed Possum GST 1–1, indicated that it possessed similar catalytic activity and structural homology with isoenzymes belonging to the alpha class of glutathione transferases. This homodimeric GST exhibited a single band with an apparent molecular mass of 25.4 kDa on sodium dodecyl sulphate-polyacrylamide gels and an apparent pI of 9.8. Inhibition studies demonstrated that Possum GST 1–1 displays binding affinity for a range of inhibitors similar to that shown by alpha class GSTs purified from other mammals. Immunoblot analysis demonstrated immuno-cross reactivity between Possum GST 1–1 and antisera raised against human alpha GST, while this GST did not cross-react with antisera raised against human mu and pi GST. N-terminal sequencing of purified Possum GST 1–1 revealed that the N-terminus of the protein is chemically blocked. Sequence analysis of three internal peptide sequences demonstrated homology with mammalian alpha GSTs. Of particular interest is the significant substrate specificity that Possum GST 1–1 displays with both organic and inorganic hydroperoxides. It is proposed that this substrate specificity is an evolutionary adaptation to a diet high in potentially toxic plant allelochemicals.

Drosophila Glutathione S-Transferase D27: Functional Analysis of Two Consecutive Tyrosines near the N-Terminus

The Drosophila glutathione S-transferase D27 (GST D27) has been purified and characterized after direct expression of the intronless gstD27 gene in E. coil. The GST D27 has both conjugation activity against the common substrate 1-chloro-2,4-dinitrobenzene and peroxidase activity against cumene hydroperoxide. Its pH optimum is 8.5 in 0.125 M bis-tris propane buffer at 22°C. It is more thermal labile than the human GST121. The GST D27 has two tyrosines at positions 3 and 4. Both of them appear to be important but neither of them is essential for the enzyme activity. Thus, other residues may also participate in catalysis. The two tyrosines of GST D27 could also be important in binding to GSH or S-hexyl GSH. Results from in vitro biochemical analyses were confirmed by the in vivo activity-based CDNB growth inhibition analyses. Our results clearly indicate that the Drosophila GST D isozymes are different from any of the known mammalian GSTs.

2,4-Dichlorophenoxyacetic Acid and Related Chlorinated Compounds Inhibit Two Auxin-Regulated Type-III Tobacco Glutathione S-Transferases.

Two auxin-inducible glutathione S-transferase (GST, EC 2.5.1.18) isozymes from tobacco (Nicotiana tabacum, White Burley) were partially characterized. GST1-1 and GST2-1 are members of a recently identified new type of plant GST isozymes that we will here refer to as type III. Both enzymes were active, with 1-chloro-2,4-dinitrobenzene as a substrate, when expressed in bacteria as fusion proteins. The apparent Km for 1-chloro-2,4-dinitrobenzene was found to be 0.85 [plus or minus] 0.25 mM for GST1-1 and 0.20 [plus or minus] 0.15 mM for GST2-1. The apparent Km for glutathione was similar for both enzymes, 0.40 [plus or minus] 0.15 mM. The in vitro activity of both enzymes could be inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic acid, with an apparent Ki of 80 [plus or minus] 40 [mu]M for GST1-1 and 200 [plus or minus] 100 [mu]M for GST2-1. The GST1-1 was also inhibited by structurally related substances, such as 2,4-dichlorobenzoic acid, with a roughly similar Ki. The nonchlorinated structures benzoic acid and phenoxyacetic acid did not inhibit. p-Chloroisobutyric acid, or clofibric acid, an auxin-transport inhibitor, was found to be an active inhibitor as well. The strongest inhibitor identified, however, was a phenylacetic acid derivative, ethacrynic acid, which showed an apparent Ki of 5 [plus or minus] 5 [mu]M for both enzymes. This substance is a known inducer as well as a substrate of specific mammalian GSTs. The results presented here indicate that the type III plant GSTs might be involved in the metabolism or transport of chlorinated substances that are structurally related to auxins. The possibility that auxins are endogenous ligands or substrates for GSTs is discussed.

Biochemical analysis of recombinant glutathione S-transferase of Fasciola hepatica

Four cDNAs encoding GST (rGST1, rGST7, rGST47 and rGST51) of Fasciola hepatica were expressed in Escherichia coli and the rGST proteins purified for biochemical analyses. The rGST proteins are 95% pure as indicated by Coomassie staining of proteins separated by SDS-PAGE. Molecular sieving by HPLC infers that, like the native protein, the rGST proteins form homodimers under non-denaturing conditions. The rGST proteins are recognised by antisera raised to the native GST of F. hepatica. All four rGST proteins from F. hepatica actively conjugate glutathione to the universal substrate, 1-chloro-2,4-dinitrobenzene. The activity of the rGSTs was also measured for substrates which have been shown to have partial specificity for the Alpha, Mu or Pi classes of mammalian GSTs ( trans-4-phenyl-3-buten-2-one, ethacrynic acid), for substrates known to be products of lipid peroxidation ( trans-2-nonenal, trans,trans-2,4-decadienal) and for epoxy-3-( p-nitrophenoxy)-propane (EPNP), a known substrate for the theta class of GST. No rGST were active with EPNP. rGST47 and 51 showed activity with the other four substrates. rGST7 was active with three substrates whereas rGST1 showed relatively low activity with all substrates except trans,trans-2,4-decadienal. The sensitivity of the rGST activity to inhibition by the GST inhibitors triphenyltin chloride and bromosulphophthalein also varied among the rGSTs with rGST1 showing a 800-fold difference in sensitivity between the inhibitors. These results show that F. hepatica expresses a family of GST isoenzymes which exhibit unique substrate and inhibitor profiles.

Glutathione S-transferases from the gastrointestinal nematode Heligmosomoides polygyrus and mammalian liver compared

Glutathione S-transferases have been partially characterised from the gastrointestinal nematode Heligmosomoides polygyrus. Two major subunit families were purified (24 and 23 kDa) with N-terminal homology to the mammalian Alpha family. Four dimeric forms of GST were purified from the nematode by glutathione-affinity chromatography, two major enzymes (pI 8.1, 5.0) and two minor forms (pI 5.8, 5.3). The purified GST pool could neutralize model and lipid peroxides via peroxidase activity but not peroxidation derived reactive carbonyls via glutathione transferase activity. Antisera raised to the pooled nematode GSTs appeared to recognize other Strongylida GSTs more strongly on Western blotting compared to mammalian GSTs.