GST A1-1 Research Articles

Determination of tetrahydrothiophene formation as a probe of in vitro busulfan metabolism by human glutathione S-transferase A1-1: use of a highly sensitive gas chromatographic–mass spectrometric method

A method for the sensitive determination of tetrahydrothiophene (THT) in cytosolic incubation mixtures was developed. Busulfan conjugation with glutathione was predominantly catalysed by glutathione S-transferase A1-1 (GST A1-1) and THT was released from the primary metabolite by alkalization. After liquid–liquid extraction using n-pentane separation and quantification of the product was performed by gas chromatography with a mass-selective detector. The method showed good sensitivity, accuracy and reproducibility with a detection limit of 2 ng ml −1 and a limit of quantification of 5 ng ml −1. The suitability of the method is shown for enzyme kinetic studies in human liver cytosol as well as for determination of GST A1-1 activity.

Human glutathione transferase A4-4 crystal structures and mutagenesis reveal the basis of high catalytic efficiency with toxic lipid peroxidation products

The oxidation of lipids and cell membranes generates cytotoxic compounds implicated in the etiology of aging, cancer, atherosclerosis, neurodegenerative diseases, and other illnesses. Glutathione transferase (GST) A4-4 is a key component in the defense against the products of this oxidative stress because, unlike other Alpha class GSTs, GST A4-4 shows high catalytic activity with lipid peroxidation products such as 4-hydroxynon-2-enal (HNE). The crystal structure of human apo GST A4-4 unexpectedly possesses an ordered C-terminal α-helix, despite the absence of any ligand. The structure of human GST A4-4 in complex with the inhibitor S-(2-iodobenzyl) glutathione reveals key features of the electrophilic substrate-binding pocket which confer specificity toward HNE. Three structural modules form the binding site for electrophilic substrates and thereby govern substrate selectivity: the β1-α1 loop, the end of the α4 helix, and the C-terminal α9 helix. A few residue changes in GST A4-4 result in α9 taking over a predominant role in ligand specificity from the N-terminal loop region important for GST A1-1. Thus, the C-terminal helix α9 in GST A4-4 provides pre-existing ligand complementarity rather than acting as a flexible cap as observed in other GST structures. Hydrophobic residues in the α9 helix, differing from those in the closely related GST A1-1, delineate a hydrophobic specificity canyon for the binding of lipid peroxidation products. The role of residue Tyr212 as a key catalytic residue, suggested by the crystal structure of the inhibitor complex, is confirmed by mutagenesis results. Tyr212 is positioned to interact with the aldehyde group of the substrate and polarize it for reaction. Tyr212 also coopts part of the binding cleft ordinarily formed by the N-terminal substrate recognition region in the homologous enzyme GST A1-1 to reveal an evolutionary swapping of function between different recognition elements. A structural model of catalysis is presented based on these results.

Benzoic acid derivatives induce recovery of catalytic activity in the partially inactive Met208Lys mutant of human glutathione transferase A1-1

Human glutathione transferase A1-1 (GST A1-1) is a detoxifying enzyme catalyzing the conjugation of glutathione with a variety of hydrophobic, electrophilic substrates. When the role of the hydrophobic substrate-binding site residue Met208 was investigated by random mutagenesis, introduction of charged amino acid residues had the greatest deleterious effect on enzyme activity. However, in the lysine mutant some of the lost activity could be regained by the addition of a benzoic acid derivative to the reaction mixture. The activating molecule has now been optimized such that all activity is recovered. The most potent activator, 4-propylbenzoic acid, has been used in studies of the mechanism behind the activation. A heterodimeric species of GST A1-1, containing only one activatable subunit, has been constructed. The heterodimer shows a strictly additive activation curve when compared to its parental forms, indicating that the activation is not due to co-operativity between the subunits. Furthermore, a novel electrophilic substrate, 4-chloro-3,5-dinitrobenzoic acid, with a carboxylate group expected to interact with residue 208 gives a higher kcat value with the lysine mutant than with wild-type GST A1-1. All results obtained in the here support the view that the positive charge introduced into the lysine mutant adversely affects the structure of the C-terminal helix of this enzyme, preventing it from adopting the conformation needed for full activity. The negatively charged carboxylate group of the activator probably neutralizes the positive charge of the side-chain amino group and thereby restores the substrate-binding site to a form that is favorable for the catalytic function.

Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S-transferase A1-1: a fluorescence spectroscopic analysis.

The binding interactions between dimeric human class alpha glutathione S-transferase A1-1 (GST A1-1) and aflatoxin B1 or sulphobromophthalein (BSP) were characterised. Aflatoxin B1 binds to GST A1-1 with a stoichiometry of 1.1 mol/mol of dimeric enzyme. The binding interaction, which can be described by a hyperbolic saturation isotherm (Kd = 8+/-2 microM), does not induce major structural changes in the enzyme, nor does it inhibit enzymatic activity. The average distance between the single tryptophan residue (Trp20) of GST A1-1 and protein-bound aflatoxin B1 was calculated to be 22.7 A by means of fluorescence resonance energy transfer. The aflatoxin-binding region, according to this calculated distance, was determined to be located in the dimer interface cleft near the crystallographic two-fold axis. Hill-plot analyses suggest that a positive co-operative interaction exists between BSP and the dimeric GST A1-1 (h = 1.6+/-0.1; K' = 14+/-0.6 microM). The binding of BSP induces a conformational change in the enzyme which is accompanied by a decrease in the molecular flexibility and in the solvent-accessible properties of the enzyme's Trp20 residue. Site-directed mutagenesis of Trp20 (Trp20-->Phe) confirms that this residue is situated in the binding environment and although it is not essential for BSP binding, it is involved in the interaction. Furthermore, the structural change associated with BSP binding alters the hyperbolic character of the glutathione saturation curve. This indicates that there may also be a cooperative interaction between glutathione and BSP or that BSP binding induces asymmetric functioning of the two enzyme subunits so that they become unequal in catalytic activity.

Phospholipid hydroperoxide glutathione peroxidase activity of human glutathione transferases.

Human glutathione transferases (GSTs) from Alpha (A), Mu (M) and Theta (T) classes exhibited glutathione peroxidase activity towards phospholipid hydroperoxide. The specific activities are in the order: GST A1-1>GST T1-1>GST M1-1>GST A2-2>GST A4-4. Using a specific and sensitive HPLC method, specific activities towards the phospholipid hydroperoxide,1-palmitoyl-2-(13-hydroper oxy-cis-9, trans-11 -octadecadienoyl)-l-3-phosphatidylcholine (PLPC-OOH) were determined to be in the range of 0.8-20 nmol/min per mg of protein. Two human class Pi (P) enzymes (GST P1-1 with Ile or Val at position 105) displayed no activity towards the phospholipid hydroperoxide. Michaelis-Menten kinetics were followed only for glutathione, whereas there was a linear dependence of rate with PLPC-OOH concentration. Unlike the selenium-dependent phospholipid hydroperoxide glutathione peroxidase (Se-PHGPx), the presence of detergent inhibited the activity of GST A1-1 on PLPC-OOH. Also, in contrast with Se-PHGPx, only glutathione could act as the reducing agent for GST A1-1. A GST A1-1 mutant (Arg15Lys), which retains the positive charge between the GSH- and hydrophobic binding sites, exhibited a decreased kcat for PLPC-OOH but not for CDNB, suggesting that the correct topography of the GSH site is more critical for the phospholipid substrate. A Met208Ala mutation, which gives a modified hydrophobic site, decreased the kcat for CDNB and PLPC-OOH by comparable amounts. These results indicate that Alpha, Mu and Theta class human GSTs provide protection against accumulation of cellular phospholipid hydroperoxides.

Human glutathione transferase A4-4: an alpha class enzyme with high catalytic efficiency in the conjugation of 4-hydroxynonenal and other genotoxic products of lipid peroxidation.

A sequence encoding a novel glutathione transferase, GST A4-4, has been identified in a human fetal brain cDNA library. The protein has been produced in Escherichia coli after optimization of the codon usage for high-level heterologous expression. The dimeric protein has a subunit molecular mass of 25704 Da based on the deduced amino acid composition. Human GST A4-4 is a member of the Alpha class but shows only 53% amino acid sequence identity with the major liver enzyme GST A1-1. High catalytic efficiency with 4-hydroxyalkenals and other cytotoxic and mutagenic products of radical reactions and lipid peroxidation is a significant feature of GST A4-4. The kcat/Km values for 4-hydroxynonenal and 4-hydroxydecenal are > 3 x 10(6) M-1. s-1, several orders of magnitude higher than the values for conventional GST substrates. 4-Hydroxynonenal and other reactive electrophiles produced by oxidative metabolism have been linked to aging, atherosclerosis, cataract formation, Parkinson's disease and Alzheimer's disease, as well as other degenerative human conditions, suggesting that human GST A4-4 fulfills an important protective role and that variations in its expression may have significant pathophysiological consequences.

Mechanism-based phage display selection of active-site mutants of human glutathione transferase A1-1 catalyzing SNAr reactions.

A library of active-site mutants has been constructed by targeting selected amino acid residues in human glutathione transferase (GST) A1-1 for random mutagenesis. The mutated residues are suitably positioned for interaction with the second, electrophilic substrate, in particular chloronitrobenzene derivatives undergoing SNAr reactions. DNA representing the GST A1-1 mutant library was fused with DNA encoding gene III protein, a component of the coat of filamentous phage. Phage display was used for affinity selection of GST A1-1 mutants with altered catalytic properties. The affinity ligand used was the sigma-complex of 1,3,5-trinitrobenzene and glutathione immobilized to Sepharose. The complex was designed to mimic the transition state of SNAr reactions catalyzed by GSTs. The selection system is based on the combination of affinity for the sigma-complex as well as the ability to promote its formation, thus mimicking two salient features of the assumed catalytic mechanism for the SNAr reactions. Many of the GST A1-1 mutants selected and analyzed contained an aromatic amino acid residue in one of the mutated positions, suggesting favorable interactions with the trinitrocyclohexadienate moiety of the affinity ligand. A mutant C36 was selected for more detailed studies. Its catalytic efficiency with several chloronitrobenzene substrates was 20-90-fold lower than that of wild-type GST A1-1, but fully comparable to naturally evolved GSTs of different classes, providing a 10(5)-fold rate enhancement over the uncatalyzed reaction. In the conjugation of ethacrynic acid, a Michael addition reaction, mutant C36 was 13-fold more efficient than the wild-type enzyme. Within experimental error, the quotient between the KF values for wild-type GST A1-1 and mutant C36 is the same as that between the kcat/KM values determined with 1-chloro-2,4-dinitrobenzene for the two enzyme forms. This result indicates that sigma-complex formation is rate-limiting for the catalyzed reaction. Thus, the principle of transition-state stabilization as a component of catalysis has been successfully exploited in affinity selection of catalytically competent GST A1-1 mutants. This mechanism-based procedure also selects for the ability to promote sigma-complex formation, and serves as a probe of the catalytic mechanism.

Characterization of a marsupial glutathione transferase, a class Alpha enzyme from Brown Antechinus ( Antechinus stuartii)

The major form of glutathione transferase from the marsupial Antechinus stuartii has been purified and characterized as an Alpha class enzyme (Ast GST A1-1) with distant sequence relationships to other class Alpha sublines, compatible with the early origin of marsupials. Amino acid replacements toward the closest enzyme characterized (chicken, form A3) involve no less than 79 positions (36%). At the active site, as deduced from comparisons with the known tertiary structure of the corresponding human enzyme, over half of the residues (8 of 15) ascribed to substrate binding interactions are exchanged although the general character of that site is conserved, while only 1 of 11 positions ascribed to interactions with GSH is exchanged. Class variability and species variability appear to coincide, with divergent segments centering around positions 33–49, 103–130 and 205–222. The pattern is reminiscent of that in similarly multiple MDR alcohol dehydrogenases. Both these enzyme families involved in cellular defense reactions have diverged considerably.

Stereoselective Conjugation of Prostaglandin A2 and Prostaglandin J2 with Glutathione, Catalyzed by the Human Glutathione S-Transferases A1-1, A2-2, M1a-1a, and P1-1

Prostaglandins containing an alpha,beta-unsaturated keto group, such as prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2), inhibit cell proliferation. These cyclopentenone prostaglandins may be conjugated with GSH chemically or enzymatically via glutathione S-transferases, and this has been suggested to result in inhibition of the antiproliferative mode of action. In the present study, the role of the major human GSTs in the conjugation of PGA2 and PGJ2 with GSH was investigated with purified enzymes, i.e., the Alpha-class enzymes GST A1-1 and GST A2-2, the Mu-class enzyme GST M1a-1a, and the Pi-class enzyme GST P1-1. The GSH conjugates were separated from the parent compound by HPLC and identified by fast atom bombardment mass spectrometry and 1H-NMR. Two GSH conjugates were found for both PGA2 and PGJ2, the R- and S-GSH conjugates of both prostaglandins. Incubation experiments with PGA2 and PGJ2 (70-600 microM) clearly showed the role of individual GSTs in the conjugation of PGA2 and PGJ2. Compared to the chemical reaction, enzyme activities towards PGA2 were up to 5.4 times as high (GSTA1-1) at the lowest concentration (70 microM), while at the highest concentration (600 microM) enzyme activities were up to 3.0 times as high (GST P1-1). For PGJ2, enzyme activities were up to 4.3 (GSTM1a-1a, 70 microM) and up to 3.1 (GSTM1a-1a, 600 microM) times as high. As expected, similar amounts of the R- and S-conjugates of both prostaglandins were found in the chemical reaction. Striking stereoselectivities in conjugating activities were observed for GST A1-1 and GST P1-1. GST A1-1 favors the formation of the R-GSH conjugates of both prostaglandins. GST P1-1 showed a clear selectivity with regard to the formation of the S-GSH conjugate of PGA2. However, this selectivity was not found for the formation of the S-GSH conjugate of PGJ2. GSTM1a-1a showed no stereoselectivity with regard to the GSH conjugation of both PGA2 and PGJ2. GSTA2-2 only showed some minor formation of the R-GSH conjugate of PGJ2. The possible implications of the observed stereoselectivity on the effects of PGA2 and PGJ2 are discussed.

Glutathione S-transferase A1-1-catalysed conjugation of bay and fjord region diol epoxides or polycyclic aromatic hydrocarbons with glutathione.

Mammalian metabolism of polycyclic aromatic hydrocarbons results in the formation of vicinal diol epoxides (existing as enantiomeric pairs of two diastereomers) considered as important ultimate carcinogens if the oxirane ring is located in a bay or fjord region of the parent hydrocarbon. In the present study, individual stereoisomers of the bay region diol epoxides of chrysene, dibenz[a,h]-anthracene and benzo[a]pyrene, as well as of the fjord region diol epoxides of benzo[c]phenanthrene, benzo[c]chrysene and benzo[g]chrysene, have been incubated with glutathione (GSH) in the presence or absence of human glutathione S-transferase isoenzyme GST A1-1, a class Alpha enzyme. The formation of GSH conjugates was determined and quantified by HPLC. The results demonstrate that the GST A1-1 isoenzyme catalyzes the formation of GSH conjugates of all diol epoxides tested, although a marked variation in catalytic efficiency (>20-fold) was observed. With both bay and fjord region anti-diol epoxides a significant preference for conjugation of the enantiomer with the R configuration at the benzylic position of the oxirane ring was noted. Among the syn diastereomers of the fjord region diol epoxides a similar substrate enantioselectivity was noted, i.e. the enantiomer with the corresponding R configuration was again preferentially conjugated. In contrast, for the bay region syn-diol epoxides this substrate selectivity was reversed, resulting in a preference for the enantiomer with the S configuration. The chemically more reactive syn diastereomers were in general better substrates for GST A1-1 than the corresponding anti diastereomers. However, a comparison between different diol epoxide diastereomers revealed no obvious correlation between chemical reactivity of the compounds and catalytic efficiencies. Furthermore, no significant correlation between diol epoxide lipophilicity and catalytic efficiency was observed. It is suggested that stereochemical factors, including the size and the geometry of the aromatic ring system and the preferred conformation of the diol epoxide, are involved as the major determinant for the rate of catalysis by GST A1-1.

Involvement of the carboxyl groups of glutathione in the catalytic mechanism of human glutathione transferase A1-1.

The present study proposes the participation of both carboxylate groups of the glutathione molecule as functional entities in the catalytic apparatus of human glutathione transferase (GST) A1-1. Functional studies in combination with structural data provide evidence for the alpha-carboxylate of the Glu residue of glutathione acting as a proton acceptor in the catalytic mechanism. The Glu carboxylate is hydrogen-bonded to a protein hydroxyl group and a main-chain NH, as well as to a water molecule of low mobility in the active site region. The Glu alpha-carboxylate of glutathione is bound in a similar manner to the active sites of mammalian glutathione transferases of classes Alpha, Mu, and Pi, for which three-dimensional structures are known. Mutation of the hydroxyl group that is hydrogen-bonded to the alpha-carboxylate of the Glu residue of glutathione (Thr68->Val) caused a shift of the pH dependence of the enzyme-catalyzed reaction, suggesting that the acidic limb of the pH-activity profile reflects the ionization of the carboxylate of the Glu residue of glutathione. The second carboxylate group of glutathione, which is part of its Gly residue, interacts with two Arg side chains in GST A1-1. One of these residues (Arg45) may influence an ionic interaction (Arg221/Asp42), which appears to contribute to binding of the second substrate by fixing the C-terminal alpha-helix as a lid over the active site. Removal of the Gly residue from the glutathione molecule caused a 13-fold increase in the KM value for the electrophilic substrate. Thus, the Gly carboxylate of glutathione, by way of influencing the topology of the active site, contributes to the binding of the second substrate of the enzyme. Consequently, the glutathione molecule has several functions in the glutathione transferase catalyzed reactions, not only as a substrate providing the thiol group for different types of chemical reactions but also as a substrate contributing a carboxylate that acts as a proton acceptor in the catalytic mechanism and a carboxylate that modulates binding of the second substrate to the enzyme.

Rational modulation of the catalytic activity of A1-1 glutathione S-transferase: evidence for incorporation of an on-face (pi...HO-Ar) hydrogen bond at tyrosine-9.

The alpha-, pi-, and mu-class glutathione S-transferases utilize a hydrogen bond between a conserved tyrosine and glutathione (GSH) to stabilize the nucleophilic thiolate anion, as Tyr-OH...-SG. This hydrogen bond is critical for efficient detoxication catalysis. The detailed structure of this hydrogen bond, however, is controlled by active site features which are not conserved across class boundaries. The alpha-class GST A1-1 has a cluster of aromatic residues on one side of the ring of the catalytic tyrosine, Tyr-9. Also, a hydrophobic Met-16 side chain is packed against the edge of the ring of Tyr-9. Molecular modeling and ab initio calculations suggested that substitution of Phe-220 with tyrosine could generate an aromatic on-face hydrogen bond (pi...HO-Ar) between the ring of Tyr-9 and the hydroxyl group of Tyr-220, and this would lower the pKa of enzyme-bound GSH. Therefore, Phe-220 was replaced by Tyr in the rat A1-1 isozyme. Also, Met-16 was replaced by Thr in order to investigate the effect of a hydrogen bond donor at the Tyr-9 ring edge. UV spectroscopic titration of GST.GSH and steady-state kinetic analysis indicate that substitution of Tyr at Phe-220 results in a decrease of the pKa of the cofactor, whereas substitution of Met-16 with Thr results in an increase of this pKa. Also, the pKa of Tyr-9 in the absence of substrates was determined directly by fluorescence titration. Substitutions F220Y and M16T resulted in a decrease of 0.5 pKa unit and an increase of 0.6 pKa unit, respectively. Together, these results indicate that a weak hydrogen bond between the engineered Tyr-220 side chain and the aromatic ring face of the catalytic Tyr-9 decreases the pKa of GSH and Tyr-9, and this alters the pH dependence of the enzymatic reaction.

Generation of a Ni(II) binding site by introduction of a histidine cluster in the structure of human glutathione transferase A1-1.

Two mutant forms of human glutathione transferase (GST) A1-1 with affinity for metal ions were constructed by introduction of His residues by site-directed mutagenesis. A mutant, 2-His, contained the mutations Lys84Gln, Asp85His and Glu88His, and another, 5-His, contained the mutations Tyr79His, Asn80His, Lys84His, Asp85His and Glu88His. The mutant proteins were obtained in good yields (40-150 mg per 3 l culture) by heterologous expression in Escherichia coli. The mutant enzymes possessed novel binding affinities for Ni(II) and Zn(II) ions, as demonstrated by immobilized metal ion affinity chromatography. The mutant with two novel His residues (2-His mutant) did not bind as tightly to immobilized Ni(II) as did the mutant with five novel His residues (5-His mutant). When tested for affinity to immobilized Zn(II), only the 5-His mutant remained bound to the column. The affinity of the 5-His mutant for Ni(II) ions in solution was determined by binding experiments in an aqueous polymeric two-phase system. Analysis of the binding curve showed two binding sites per enzyme subunit and a dissociation constant of 6.7 +/- 1.6 mu M. The kinetic constants kcat, Km and kcat/Km for the reaction with glutathione and 1-chloro-2,4-dinitrobenzene were determined by steady-state kinetic analysis and the parameter values for the mutant forms were found to be indistinguishable from those obtained for the wild-type GST A1-1. The differences in surface charge in the mutant proteins as compared with the wild-type enzyme did not alter the pH dependence of kcat. The results provide an alternative method for purification of fully active recombinant GST A1-1 by the introduction of novel metal binding sites. The data also showed that two His residues are sufficient for Ni(II) binding.

Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.

Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases.

Radiation and chemical reactions that give rise to free radicals cause the formation of highly cytotoxic base propenals, degradation products of DNA. Human glutathione transferases (GSTs; RX:glutathione R-transferase, EC 2.5.1.18) of classes Alpha, Mu, and Pi were shown to promote the conjugation of glutathione with base propenals and related alkenes. GST P1-1 was particularly active in catalyzing the reactions with the propenal derivatives, and adenine propenal was the substrate giving the highest activity. The catalytic efficiency of GST P1-1 with adenine propenal (kcat/Km = 7.7 x 10(5) M-1.s-1) is the highest so far reported with any substrate for this enzyme. In general, GST A1-1 and GST M1-1, in contrast to GST P1-1, were more active with 4-hydroxyalkenals (products of lipid peroxidation) than with base propenals. The adduct resulting from the Michael addition of glutathione to the alkene function of one of the base propenals (adenine propenal) was identified by mass spectrometry. At the cellular level, GST P1-1 was shown to provide protection against alpha, beta-unsaturated aldehydes. GST P1-1 added to the culture medium of HeLa cells augmented the protective effect of glutathione against the toxicity of adenine propenal and thymine propenal. No protective effect of the enzyme was observed in the presence of the competitive inhibitor S-hexylglutathione. GST P1-1 introduced into Hep G2 cells by electroporation was similarly found to increase their resistance to acrolein. The results show that glutathione transferases may play an important role in cellular detoxication of electrophilic alpha, beta-unsaturated carbonyl compounds produced by radical reactions, lipid peroxidation, ionizing radiation, and drug metabolism.

Cell cycle dependent sensitivity of human melanoma cells to melphalan is correlated with the activity and cellular concentration of glutahione transferases

Glutathione transferases (GSTs) are enzymes involved in the resistance of tumor cells to bifunctional alkylating cytostatic drugs. We investigated the melphalan sensitivity together with activity and cellular concentration of GST isoenzymes of human melanoma cell line RPMI 8322 in different phases of the cell cycle. By centrifugal elutriation three cell fractions containing different proportions of cells in the G1 phase were isolated. Melphalan sensitivity was estimated by the colony formation assay. The cell fraction with the largest proportion of G1 cells was more sensitive to the drug than the fractions enriched in S and G2 cells. The GST activity of the cell fractions was measured with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate and the concentrations of GST P1-1, GST M1-1 and GST A1-1 were quantitated by use of isoenzyme-specific ELISA. The results show that there were less GST activity and lower GST P1-1 and A1-1 concentrations in the G1 cell enriched fraction, demonstrating a cell cycle dependence of GST expression. Thus, the cell fraction most sensitive to melphalan had the highest proportion of G1 cells and displayed the lowest GST activity, suggesting that the cell cycle dependent sensitivity to melphalan may at least partially depend on the expression of GSTs.

Isoenzyme-specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines

Enzyme-linked immunoassays (ELISAs) based on the double-antibody sandwich technique have been developed for the quantitative analysis of the major human cytosolic class Pi, Mu and Alpha glutathione transferases (GSTs). The procedures were optimized with respect to antibody concentration for coating of plates as well as other parameters in order to achieve high sensitivity and accuracy. No cross-reactivity was detected between members of the three different classes of GSTs or among the Mu class GSTs M2-2, M3-3 and M4-4 with the ELISA for GST M1-1. The ELISAs have been applied to establish the cytosolic GST profiles of 10 cell lines and to monitor the plasma GST levels in cancer patients. The results revealed that the class Pi GST was the dominant isoenzyme in six (LS 174T, HCT-8, Hu 549 Pat, K-562, U-937 and Hu 549) out of nine tumor cell lines and immortalized hepatocytes (Chang Liver). The isoenzymes A1-1 and M1-1 were determined to be the major GST components in Hep G2 and HeLa cells, respectively. In a clinical study, the majority of the patients with urinary bladder cancer were found to have increased plasma levels of both GST A1-1 and GST P1-1 (10/15), while patients with renal cancer frequently showed increases only in GST P1-1 (5/8). The results demonstrate that the ELISAs are suitable for analyzing GST phenotypes in both normal and tumor cells and in monitoring plasma levels of GSTs in cancer patients.

Co-variation of glutathione transferase expression and cytostatic drug resistance in HeLa cells: establishment of class Mu glutathione transferase M3-3 as the dominating isoenzyme.

Qualitative and quantitative analyses of glutathione, glutathione transferases (GSTs) and other glutathione-linked enzymes in HeLa cells have been made in order to study their significance in cellular resistance to electrophilic cytotoxic agents. The cytosolic concentrations of three GSTs, GST M1-1 (53 +/- 9 ng/mg of cytosolic protein), GST P1-1 (11 +/- 3 ng/mg) and GST A1-1 (1.1 +/- 0.4 ng/mg) were quantified by isoenzyme-specific enzyme-linked immunoassays. Electrophoretic analysis and immunoblotting demonstrated another component, GST M3-3, which was identified by amino acid sequence analysis. GST M3-3 was quantified (1550 +/- 250 ng/mg) by slot-blot immunoanalysis and was the most abundant GST in HeLa cells. An additional cytosolic 13 kDa protein with high affinity for immobilized glutathione or S-hexyglutathione was found to be identical with a macrophage migration-inhibitory factor, previously identified as a lymphokine. Cells grown in roller bottles (HR) rather than in ordinary culture flasks contain a significantly lower concentration of all the GSTs and were found to be more sensitive to the cytostatic agents doxorubicin (2.3-fold), cisplatin (1.7-fold) and melphalan (1.4-fold). The cytosolic concentrations of glutathione reductase and glyoxalase I were also lower in HR cells, whereas the total glutathione concentration was unchanged and the glutathione peroxidase activity was increased. The results indicate that GSTs contribute to the cellular resistance phenotype.

Glutathione conjugation of trans-3,4-dihydroxy 1,2-epoxy 1,2,3,4-tetrahydrobenzo[c]phenanthrene isomers by human glutathione transferases.

Each of the four stereoisomers of trans-3,4-dihydroxy 1,2-epoxy 1,2,3,4-tetrahydrobenzo[c]phenanthrene [(+)- and (-)-anti-BPhDE and (+)- and (-)-syn-BPhDE] has been incubated with the human glutathione transferase (GST) isoenzymes GST A1-1, GST M1-1 and GST P1-1, representing class alpha, mu and pi respectively, and glutathione (GSH). The conjugates formed were analyzed by HPLC and the results demonstrate that all GST isoenzymes catalyze the formation of GSH conjugates of all BPhDE isomers. However, a marked variation in catalytic efficiencies was observed (0.122-1.28/mM/s). These values are considerably lower than those previously estimated for the bay-region diol epoxides of benzo[a]pyrene (B[a]P) and human GSTs. The (+)-syn and (-)-anti-BPhDE (1R,2S-epoxide absolute configuration) were in general better substrates than the corresponding 1S,2R-epoxides. In accordance with previous observations with the diolepoxides of B[a]P, GST P1-1 was highly selective towards the BPhDE isomer with 4R,3S-diol 2S,1R-epoxide absolute configuration, i.e. (-)-anti-BPhDE, whereas GST A1-1 and M1-1 preferentially catalyzed the conjugation of (+)-syn-BPhDE (4R,3S-diol 2R,1S-epoxide absolute configuration). Overall, the most active isoenzyme was GST A1-1. Analysis by NMR spectroscopy of the GSH conjugates of BPhDE demonstrate that the reaction with GSH generally takes place by trans-addition of the thiol group at the benzylic C-1 carbon. The low catalytic efficiencies of human GSTs with BPhDE as compared to diolepoxides of B[a]P may be explained in part by the more crowded bay-region and substantially lower chemical reactivity (e.g. delta Edeloc/beta) of the former compounds.