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PubMed2004

Molecular cloning and characterization of a novel glutathione S-transferase gene induced by light stimulation in the protozoan Blepharisma japonicum.

A cDNA clone that is inducible by light stimulation was cloned by a differential screening method from a cDNA library of the protozoan Blepharisma japonicum, and the light-dependent expression was checked by semi-quantitative reverse transcription polymerase chain reaction analysis. Sequence analysis showed that the cDNA encodes a glutathione S-transferase (GST) that has not been characterized in the protozoa. Multiple alignment of B. japonicum GST (BjGST1), known protozoan, and mammalian alpha-, micro-, pi-, sigma-, theta-, zeta-, kappa-, and omega-class GSTs suggested that the BjGST1 may be a novel class GST. Furthermore, highly conserved amino acid residues among the GSTs and the substrate specificity of recombinant BjGST1 showed that BjGST1 is related to alpha-, micro-, pi-, and sigma-class GSTs rather than the other class of GSTs.

Amino Acid Sequence↗
PubMedDate not supplied

Cloning, expression and characterization of an insect class I glutathione S-transferase from Anopheles dirus species B.

Insect class I glutathione S-transferases (GSTs) were expressed from cDNA obtained from larvae of the Thai malaria vector. Anopheles dirus in a PCR RACE (rapid amplification of cDNA ends) reaction using a primer to the conserved N-terminal region of An. gambiae class I GSTs and a synthetic oligo d(T)-adaptor primer. Seven different plasmids, resulting from sub-cloning of an original single 0.7 Kb PCR band, were picked at random and sequenced. Four of these were clearly GSTs on the basis of putative amino acid sequence conservation. All the sequences had a conserved N-terminal region, but were highly divergent at the C-terminus. The variability in the PCR products suggests that there is a high level of GST class I isoenzyme variability in larval An. dirus. One of the subclones from the PCR reaction contained a full coding region of the cDNA for GST. This had a putative amino acid sequence which was 76 and 91% identity to the An. gambiae GST class I, agGST 1-5 and agGST 1-6 respectively, but only 48% identity to agGST 1-2. The catalytically active enzyme, expressed in Escherichia coli, was strongly immuno-cross reactive with antisera raised against the two An. gambiae class I GSTs. The expressed enzyme was purified to homogeneity from an E. coli cell lysate by S-hexylglutathione agarose affinity chromatography. The enzyme had a high specific activity with CDNB, and also used DCNB and ethacrynic acid as substrates. In addition, it had peroxidase and DDTase activity and its activity with CDNB, was strongly inhibited by a range of organophosphorus and pyrethroid insecticides. This is consistent with the predicted role of this GST class in insecticide resistance.

Amino Acid Sequence↗
PubMed1997

Structures of class pi glutathione S-transferase from human placenta in complex with substrate, transition-state analogue and inhibitor.

BACKGROUND: Glutathione S-transferases (GSTs) are detoxification enzymes, found in all aerobic organisms, which catalyse the conjugation of glutathione with a wide range of hydrophobic electrophilic substrates, thereby protecting the cell from serious damage caused by electrophilic compounds. GSTs are classified into five distinct classes (alpha, mu, pi, sigma and theta) by their substrate specificity and primary structure. Human GSTs are of interest because tumour cells show increased levels of expression of single classes of GSTs, which leads to drug resistance. Structural differences between classes of GST can therefore be utilised to develop new anti-cancer drugs. Many mutational and structural studies have been carried out on the mu and alpha classes of GST to elucidate the reaction mechanism, whereas knowledge about the pi class is still limited. RESULTS: We have solved the structures of the pi class GST hP1-1 in complex with its substrate, glutathione, a transition-state complex, the Meisenheimer complex, and an inhibitor, S-(rho-bromobenzyl)-glutathione, and refined them to resolutions of 1.8 A, 2.0 A and 1.9 A, respectively. All ligand molecules are well-defined in the electron density. In all three structures, an additionally bound N-morpholino-ethansulfonic acid molecule from the buffer solution was found. CONCLUSIONS: In the structure of the GST-glutathione complex, two conserved water molecules are observed, one of which hydrogen bonds directly to the sulphur atom of glutathione and the other forms hydrogen bonds with residues around the glutathione-binding site. These water molecules are absent from the structure of the Meisenheimer complex bound to GST, implicating that deprotonation of the cysteine occurs during formation of the ternary complex which involves expulsion of the inner bound water molecule. The comparison of our structures with known mu class GST structures show differences in the location of the electrophile-binding site (H-site), explaining the different substrate specificities of the two classes. Fluorescence measurements are in agreement with the position of the N-morpholino-ethansulfonic acid, close to Trp28, identifying a possible ligandin-substrate binding site.

Alkanesulfonic Acids↗
PubMed1998

Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site.

BACKGROUND: Glutathione S-transferases (GSTs) comprise a multifunctional group of enzymes that play a critical role in the cellular detoxification process. These enzymes reduce the reactivity of toxic compounds by catalyzing their conjugation with glutathione. As a result of their role in detoxification, GSTs have been implicated in the development of cellular resistance to antibiotics, herbicides and clinical drugs and their study is therefore of much interest. In mammals, the cytosolic GSTs can be divided into five distinct classes termed alpha, mu, pi, sigma and theta. The human theta class GST, hGST T2-2, possesses several distinctive features compared to GSTs of other classes, including a long C-terminal extension and a specific sulfatase activity. It was hoped that the determination of the structure of hGST T2-2 may help us to understand more about this unusual class of enzymes. RESULTS: Here we present the crystal structures of hGST T2-2 in the apo form and in complex with the substrates glutathione and 1-menaphthyl sulfate. The enzyme adopts the canonical GST fold with a 40-residue C-terminal extension comprising two helices connected by a long loop. The extension completely buries the substrate-binding pocket and occludes most of the glutathione-binding site. The enzyme has a purpose-built novel sulfate-binding site. The crystals were shown to be catalytically active: soaks with 1-menaphthyl sulfate result in the production of the glutathione conjugate and cleavage of the sulfate group. CONCLUSIONS: hGST T2-2 shares less than 15% sequence identity with other GST classes, yet adopts a similar three-dimensional fold. The C-terminal extension that blocks the active site is not disordered in either the apo or complexed forms of the enzyme, but nevertheless catalysis occurs in the crystalline state. A narrow tunnel leading from the active site to the surface may provide a pathway for the entry of substrates and the release of products. The results suggest a molecular basis for the unique sulfatase activity of this GST.

Binding Sites↗
PubMed1998

Structures of herbicides in complex with their detoxifying enzyme glutathione S-transferase - explanations for the selectivity of the enzyme in plants.

BACKGROUND: Glutathione S-transferases (GSTs) are detoxifying enzymes present in all aerobic organisms. These enzymes catalyse the conjugation of glutathione with a variety of electrophilic compounds. In plants, GSTs catalyse the first step in the degradation of several herbicides, such as triazines and acetamides, thus playing an important role in herbicide tolerance. RESULTS: We have solved the structures of GST-I from maize in complex with an atrazine-glutathione conjugate (at 2.8 A resolution) and GST from Arabidopsis thaliana (araGST) in complex with an FOE-4053-glutathione conjugate (at 2.6 A resolution). These ligands are products of the detoxifying reaction and are well defined in the electron density. The herbicide-binding site (H site) is different in the two structures. The architecture of the glutathione-binding site (G site) of araGST is different to that of the previously described structure of GST in complex with two S-hexylglutathione molecules, but is homologous to that of GST-I. CONCLUSIONS: Three features are responsible for the differences in the H site of the two GSTs described here: the exchange of hydrophobic residues of different degrees of bulkiness; a slight difference in the location of the H site; and a difference in the degree of flexibility of the upper side of the H site, which is built up by the loop between helices alpha4 and alpha5. Taking these two structures as a model, the different substrate specificities of other plant GSTs may be explained. The structures reported here provide a basis for the design of new, more selective herbicides.

Amino Acid Sequence↗
PubMed2000

Characterization of two mu class glutathione S-transferases from guinea pig lens.

Glutathione S-transferase (GST) plays an important role in the detoxifications of foreign electrophiles. Two GSTs of class mu from guinea pig lens were purified with Sephacryl S-100 gelfiltration, S-Hexyl glutathione Agarose affinity and Q-Sepharose anion exchange chromatographies. These GSTs (GST-A and B) showed similar relative molecular masses of 22.9 and 22.5 kDa, respectively. Two protein bands which crossreacted with anti GSTYb1 (GST 3-3) were detected in lens cytosolic crude extract on Western blotting and they showed Mrs corresponding to the purified enzymes. These GSTs showed a strong resistance against H2O2, 1,2-naphthoquinone and superoxide anion consistent with the other GSTs in class mu from animal tissues.

Animals↗
PubMed2000

Mammalian class theta GST and differential susceptibility to carcinogens: a review.

Glutathione S-transferases (GSTs) are an important part of the cellular detoxification system and, perhaps, evolved to protect cells against reactive oxygen metabolites. Theta is considered the most ancient among the GSTs and theta-like GSTs are found in mammals, fish, insects, plants, unicellular algae, and bacteria. It is thought that an ancestral theta-gene underwent an early duplication before the divergence of fungi and animals and further duplications generated the variety of the other classes of GSTs (alpha, mu, phi, etc.). The comparison of the aminoacidic homologies among mammals suggests that a duplication of an ancient GST theta occurred before the speciation of mammals and resulted in the subunits GSTT1 and GSTT2. The ancestral GST theta has a dehalogenase activity towards several halogenated compounds, such as the dichloromethane. In fact, some aerobic and anaerobic methylotrophic bacteria can use these molecules as the sole carbon and energy source. The mammalian GST theta cannot sustain the growth of bacteria but still retains the dehalogenating activity. Therefore, although mammalian GST theta behaves as a scavenger towards electrophiles, such as epoxides, it acts also as metabolic activator for halogenated compounds, producing a variety of intermediates potentially dangerous for DNA and cells. For example, mice exposed to dichloromethane show a dose-dependent incidence of cancer via the GSTT1-1 pathway. Because GSTT1-1 is polymorphic in humans, with about 20% of Caucasians and 80% of Asians lacking the enzyme, the relationship between the phenotype and the incidence of cancer has been investigated extensively in order to detect GSTT1-1-associated differential susceptibility towards endogenous or exogenous carcinogens. The lack of the enzyme is related to a slightly increased risk of cancer of the bladder, gastro-intestinal tract, and for tobacco-related tumors (lung or oral cavity). More pronounced risks were found in males with the GSTT1-null genotype for brain diseases and skin basal cell carcinomas not related to sunlight exposures. Moreover, there was an increased risk of kidney and liver tumors in humans with the GSTT1-1 positive genotype following exposures to halogenated solvents. Interestingly, the liver and kidney are two organs that express the highest level of GST theta in the human body. Thus, the GSTT1-1 genotype is suspected to confer decreased or increased risk of cancer in relation to the source of exposure; in vitro studies, mostly conducted on metabolites of butadiene, confirm the protective action of GSTT1-1, whereas, thus far, experimental studies prove that the increasing risk is limited.

Amino Acid Sequence↗
PubMed2001

Crystal structures of the yeast prion Ure2p functional region in complex with glutathione and related compounds.

The [URE3] phenotype in yeast Saccharomyces cerevisiae is due to an altered prion form of Ure2p, a protein involved in nitrogen catabolism. To understand possible conformational changes at the origin of prion propagation, we previously solved the crystal structure of the Ure2p functional region [Bousset et al. (2001) Structure 9, 39-46]. We showed the protein to have a fold similar to that of the beta class of glutathione S-transferases (GSTs). Here we report crystal structures of the Ure2p functional region (extending from residues 95-354) in complex with glutathione (GSH), the substrate of all GSTs, and two widely used GST inhibitors, namely, S-hexylglutathione and S-p-nitrobenzylglutathione. In a manner similar to what is observed in many GSTs, ligand binding is not accompanied by a significant change in the conformation of the protein. We identify one GSH and one hydrophobic electrophile binding site per monomer as observed in all other GSTs. The sulfur group of GSH, that conjugates electrophiles, is located near the amide group of Asn124, allowing a hydrogen bond to be formed. Biochemical data indicate that GSH binds to Ure2p with high affinity. Its binding affects Ure2p oligomerization but has no effect on the assembly of the protein into amyloid fibrils. Despite results indicating that Ure2p lacks GST activity, we propose that Ure2p is a member of the GST superfamily that may describe a novel GST class. Our data bring new insights into the function of the Ure2p active region.

Amino Acid Sequence↗
PubMed1995

Purification, mass spectrometric characterization, and covalent modification of murine glutathione S-transferases.

Three cytosolic glutathione S-transferase (GST, EC 2.5.1.18) isozymes were purified from livers of male CD-1 mice to homogeneity using affinity chromatography and gradient elution. Isozyme molecular masses and purities were determined using electrospray ionization mass spectrometry (ESI/MS), HPLC, and electrophoretic methods. Isozymes were assigned to alpha, pi, and mu classes based on kinetic and electrophoretic properties. Molecular masses determined by mass spectrometry were 25 271.4 +/- 2, 23 478.8 +/- 2, and 25 838.4 +/- 2 Da for alpha, pi, and mu isozymes, respectively. Molecular masses for pi and mu class isozymes agreed with molecular masses derived from nucleic acid sequences, but the molecular mass of the alpha class isozyme was 42 Da greater than calculated for a reported sequence. Analysis of tryptic digests of GST-alpha using tandem mass spectrometry determined that the molecular weight discrepancy could be attributed to N-terminal acetylation. Effects of covalent attachment of 4-hydroxy-2-nonenal (HNE) on catalytic activities of each GST isozyme were investigated by incubating GSTs with HNE. The extent of HNE-protein adduct formation was determined using ESI/MS. Catalytic activity of HNE-modified GSTs toward CDNB was measured. ESI mass spectra of modified enzymes show that the extent of HNE modification to the isozymes (pi > alpha > mu) coincides with the loss of activity seen for each protein. ESI mass spectra also indicated that glutathione-protein adducts form in isozymes during incubations with HNE even though purified proteins had been dialyzed against water before incubations. Glutathionylation of isozymes increased as the concentration of HNE in the incubations was raised. Dialysis of GSTs against 2 mM S-hexylglutathione (to displace GSH) before incubations eliminated S-glutathionylation. Reactivities of these GSTs toward HNE differed and is related to the number of cysteine residues (alpha < mu < pi) within the amino acid sequence.

Animals↗
PubMed2001

Multicenter study of the frequency of contact allergy to gold.

Gold sodium thiosulfate (GSTS) is reputed to be the most reliable gold antigen, but control studies are still required. Although Koreans have more varied sources of contact with gold, such as herbal medicines with gold coatings and indwelling gold acupuncture needles, no epidemiological studies have been performed. This study examined the frequency and sources of contact allergy to gold in South Korea by a multicenter study. Patch testing with 0.5% GSTS in pet. was conducted in 255 eczema patients and 58 control subjects. Results were observed at 21 days (D) to ensure there were no undetected late reactions in 54 observed patients and 47 controls. 8 (3.1%) of the 255 patients reacted positively to GSTS, including 1 with a late reaction. 1 of the 58 controls showed a positive reaction to GSTS on D4 with a negative reaction on D14. Clinical relevance was lacking in the patients with positive reactions. Unexpectedly, the above particular sources of gold contact gave rise to few allergic reactions in our patients.

Adolescent↗
PubMed1992

Chlorambucil-monoglutathionyl conjugate is sequestered by human alpha class glutathione S-transferases.

The spontaneous reaction of 110 microM chlorambucil (4-[p-[bis(2-chloroethyl)amino]phenyl]-butanoic acid; CHB) with 5 mM GSH at 37 degrees C in physiological phosphate-buffered saline for 35 min gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl,N-2-S-glutathionylethyl]amino]phenyl]-butano ic acid; CHBSG) and the diglutathionyl derivative, 4-[p-[bis(2-S-glutathionylethyl]amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivatives: 4-[p-[N-2-chloroethyl,N-2-hydroxy-ethyl]amino] phenyl-butanoic acid (CHBOH) and 4-[p-[N-2-S-glutathionylethyl-2-hydroxyethyl]amino]phenyl]-butanoi c acid (CHBSGOH). The inclusion of approximately physiological amounts of human glutathione S-transferases (GSTs) A1-1, A2-2, P1-1, M1a-1a M3-3 or P1-1 (for nomenclature see Mannervik et al., 1992, Biochem. J., 282, 305) had little or no catalytic effect on these reactions as determined by loss of CHB. However, GTSs A1-1 and A2-2 were associated with a significant increase of CHBSG at the expense of CHBSG2 + CHBSGOH suggesting that these GTs sequestered CHBSG at the active site. This interpretation was supported by inhibition studies which showed that CHBSG was a pure competitive inhibitor of the activity of GSTs A1-1 and A2-2 towards 1-chloro-2,4-dinitrobenzene with Ki's of 1.3 and 1.2 microM respectively. GSH transferases P1-1 and M1a-1a were inhibited by CHBSG above 10 microM. Incubation of 2 microM CHB, a concentration which may be of more significance for chemotherapy, in the presence or absence of GST A1-2 (20-50 microM) showed catalysis of GSH monoconjugation equivalent to 18% of the spontaneous rate. However, the dominant effect again was the sequestration of CHBSG which reached 74.3 +/- 1.5 (SEM)% of the total reactants at 60 min compared to 28.9 +/- 0.3(SEM)% in controls. CHBSG, although possessing a potential electrophilic centre, showed no detectable alkylation of plasmid DNA but indirect evidence was obtained that it alkylated other cellular macromolecules. It is concluded that the contribution of GSTs to catalysis of CHB detoxication will depend on factors not previously considered, namely the relative molarities of CHB, CHBSG and GSTs, and the cellular capacity to excrete CHBSG to relieve product inhibition.

Chlorambucil↗
PubMed1994

Modulation of cisplatin cytotoxicity by sulphasalazine.

The efficacy of cisplatin [cis-diamminedichloroplatinum (II); DDP] is hampered by acquired or de novo resistance of malignant cells to its cytotoxic effects. We have previously reported that cisplatin resistance parallels glutathione S-transferase (GST) activity in several human small-cell lung cancer cell lines. In the presently described studies, we used sulphasalazine, an inhibitor of GSTs, to evaluate the relative role of GSTs in mediating cisplatin resistance in two human small-cell lung cancer cell lines, NCI H-69 and H-2496. The H-69 cell line, which contained relatively higher GST activity than the H-2496 cell line (317 +/- 7 vs 9 +/- 1 mU mg-1 protein respectively), also displayed a greater degree of cisplatin resistance (IC50 values of 25.0 +/- 3.9 vs 4.5 +/- 1.0 microM respectively). Western blot and Northern blot analyses of purified GSTs revealed the expression of only the pi-class GST in both cell lines. Sulphasalazine inhibited the purified GSTs (IC50 of 10 microM for H-69 and 12 microM for H-2496) from both lines in a competitive manner with similar Ki values (6.5 and 7.9 microM for the H-69 and H-2496 cell lines respectively). Cytotoxicity studies revealed that sulphasalazine increased the cytotoxicity of cisplatin towards both cell lines. Isobologram analysis showed that sulphasalazine synergistically enhanced the cytotoxicity of cisplatin towards both cell lines, the magnitude of synergy being remarkably higher in H-69 cells than in H-2496 cells. Our studies indicate that clinically achievable concentrations of sulphasalazine may be useful in modulating cisplatin resistance in malignancies with increased GST-pi content.

Carcinoma, Small Cell↗
PubMed2001

Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione.

Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.

Amino Acid Sequence↗
PubMed2001

Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae.

The sequence and cytological location of five Anopheles gambiae glutathione S-transferase (GST) genes are described. Three of these genes, aggst1-8, aggst1-9 and aggst1-10, belong to the insect class I family and are located on chromosome 2R, in close proximity to previously described members of this gene family. The remaining two genes, aggst3-1 and aggst3-2, have a low sequence similarity to either of the two previously recognized classes of insect GSTs and this prompted a re-evaluation of the classification of insect GST enzymes. We provide evidence for seven possible classes of insect protein with GST-like subunits. Four of these contain sequences with significant similarities to mammalian GSTs. The largest novel insect GST class, class III, contains functional GST enzymes including two of the A. gambiae GSTs described in this report and GSTs from Drosophila melanogaster, Musca domestica, Manduca sexta and Plutella xylostella. The genes encoding the class III GST of A. gambiae map to a region of the genome on chromosome 3R that contains a major DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] resistance gene, suggesting that this gene family is involved in GST-based resistance in this important malaria vector. In further support of their role in resistance, we show that the mRNA levels of aggst3-2 are approx. 5-fold higher in a DDT resistant strain than in the susceptible strain and demonstrate that recombinant AgGST3-2 has very high DDT dehydrochlorinase activity.

Amino Acid Sequence↗
PubMed2002

Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.

A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens.

Amino Acid Sequence↗
PubMed2002

Cloning and expression of a novel Mu class murine glutathione transferase isoenzyme.

The present study describes the cDNA cloning, expression and characterization of a novel Mu class murine glutathione transferase (GST) isoenzyme. Screening of a cDNA library from the small intestine of a female A/J mouse using consensus probes derived from Mu class murine GST genes (mGSTM1-mGSTM5) resulted in the isolation of a full-length cDNA clone of a previously unknown Mu class GST gene (designated as mGSTM7). The choice of tissue was based on our previous identification in female A/J mouse small intestine of a potentially novel Mu class GST isoenzyme. The deduced amino acid sequence of mGSTM7, which comprises of 218 amino acid residues, exhibited about 67-78% identity with other Mu class murine GSTs. Recombinant mGSTM7-7 cross-reacted with anti-(GST Mu) antibodies, but not with anti-(GST Alpha) or anti-(GST Pi) antibodies. The pI and the reverse-phase-HPLC elution profile of recombinant mGSTM7-7 were different from those of other Mu class murine GSTs. The substrate specificity of mGSTM7-7 was also different compared with other Mu class murine GSTs. Interestingly, mGSTM7 had a higher identity with the human Mu class isoenzyme hGSTM4 (87% identity and 94% similarity in the amino acid sequence) than with any of the known mouse Mu class GSTs. Specific activities of recombinant mGSTM7-7 and human GSTM4-4 were comparable towards several substrates. For example, similar to hGSTM4-4, recombinant mGSTM7-7 was poorly active in catalysing the GSH conjugation of 1-chloro-2,4-dinitrobenzene and ethacrynic acid, and lacked activity towards 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. These results suggested that hGSTM4-4 might be the human counterpart of mouse GSTM7-7. Reverse transcription-PCR analysis using mGSTM7-specific primers revealed that mGSTM7 is widely expressed in tissues of female A/J mice, including liver, forestomach, lung, kidney, colon and spleen.

Amino Acid Sequence↗
PubMed2002

Multiple isoforms of mitochondrial glutathione S-transferases and their differential induction under oxidative stress.

The mitochondrial respiratory chain, which consumes approx. 85-90% of the oxygen utilized by cells, is a major source of reactive oxygen species (ROS). Mitochondrial genetic and biosynthetic systems are highly susceptible to ROS toxicity. Intramitochondrial glutathione (GSH) is a major defence against ROS. In the present study, we have investigated the nature of the glutathione S-transferase (GST) pool in mouse liver mitochondria, and have purified three distinct forms of GST: GSTA1-1 and GSTA4-4 of the Alpha family, and GSTM1-1 belonging to the Mu family. The mitochondrial localization of these multiple GSTs was confirmed using a combination of immunoblot analysis, protease protection assay, enzyme activity, N-terminal amino acid sequencing, peptide mapping and confocal immunofluorescence analysis. Additionally, exogenously added 4-hydroxynonenal (HNE), a reactive byproduct of lipid peroxidation, to COS cells differentially affected the cytosolic and mitochondrial GSH pools in a dose- and time-dependent manner. Our results show that HNE-mediated mitochondrial oxidative stress caused a decrease in the GSH pool, increased membrane lipid peroxidation, and increased levels of GSTs, glutathione peroxidase and Hsp70 (heat-shock protein 70). The HNE-induced oxidative stress persisted for longer in the mitochondrial compartment, where the recovery of GSH pool was slower than in the cytosolic compartment. Our study, for the first time, demonstrates the presence in mitochondria of multiple forms of GSTs that show molecular properties similar to those of their cytosolic counterparts. Our results suggest that mitochondrial GSTs may play an important role in defence against chemical and oxidative stress.

Aldehydes↗
PubMed2003

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↗