Glutathione S-transferase Isoenzymes Research Articles

Immunohistochemical Demonstration of Glycoprotein 170 and Glutathione S-Transferase Isoenzymes in Normal and Neoplastic Bronchial Tissue

Background: The aim of this study was to demonstrate the content of drug-transporting and -metabolizing enzymes in primary lung cancer. Material and Methods: We investigated 20 specimens of lobectomies, bilobectomies, pneumonectomies and extended pneumonectomies of patients suffering from lung Results: In normal bronchial tissue and various primary lung tumors, we observed a high content of glycoprotein 170 (gp 170), and glutathione S-transferase (GST) placenta (π) and liver (α) types. Gp 170 and GSTπ were localized in columnar epithelium, excretory ducts, serous glands, muscles, cartilage, nerves and vessel walls. Nine of 12 squamous cell carcinomas showed a considerable quantity of gp 170, comparable with the amount of gp 170 in the proximal tubules of the kidney. All of them stained for GST π. GST α was demonstrable to a lesser extent than GSTπ. Only few of the carcinomas stained markedly positive for GSTα. There was no correlation between the quantity in normal bronchial tissue and the corresponding tumor for any enzyme studied. Conclusions: If gp 170 and GST play a role in primary chemoresistance, expecially in multiple drug resistance, our results explain the high rate of primary chemoresistance in non-small cell lung tumors.

Inhibition of rat liver cytosolic glutathione S-transferase by silybin.

1. The naturally occurring hepatoprotective compound silybin is a potent inhibitor of glutathione S-transferase isoenzymes 1-1, 2-2, 3-3, and 4-4, displaying a high degree of isoenzyme selectivity. 2. Using CDNB at a fixed concentration of 1 mM and varying the GSH concentration, silybin exhibited competitive inhibition of isoenzyme 2-2 with a Ki of 32 microM, non-competitive and predominantly non-competitive inhibition of isoenzymes 1-1 and 4-4 with Kis of 20 and 1.2 microM, respectively, and uncompetitive inhibition of isoenzyme 3-3 with a Ki of 0.2 microM. 3. With CDNB as the variable concentration substrate silybin exhibited competitive inhibition of isoenzyme 1-1 with a Ki of 8 microM, non-competitive inhibition of isoenzyme 2-2 with a Ki of 41 microM, and non-competitive inhibition of isoenzymes 3-3 and 4-4 with Kis of 0.8 and 0.5 microM, respectively.

Glutathione transferase isoenzymes in olfactory and respiratory epithelium of cattle

Glutathione transferase (GST) was investigated in the olfactory and respiratory epithelium of cattle. A significantly more abundant GST in terms of either protein amount or activity was found in the olfactory rather than in the respiratory epithelium. No apparent qualitative differences in the isoelectric focusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC profiles were noted in the reduced glutathione (GSH) affinity purified GST pool of olfactory and respiratory epithelium. Both tissues have at least six GST isoenzymes with isoelectric point values of 4.9 (peak I), 5.3 (peak II), 5.95 (peak III), 6.5 (peak IV), 7.1 (peak V) and 9.3 (peak VI). From both tissues at least seven different GST subunits can be resolved by HPLC analysis. The GST isoenzymes having pI at 5.3 and 9.3 were predominantly expressed in the olfactory than in the respiratory epithelium. These latter forms conjugate GSH efficiently with alkenals and hydroperoxides, respectively. Kinetic, immunological and structural properties, including HPLC analysis and N-terminal region amino acid sequence seem to indicate that the bovine nasal mucosa tissue in addition to a GST subunit which is orthologue to rat subunit 8 (alpha class) express tissues specific subunits.

Fluorescence characterization of Trp 21 in rat glutathione S‐transferase 1–1: Microconformational changes induced by S‐hexyl glutathione

The glutathione S-transferase (GST) isoenzyme A1-1 from rat contains a single tryptophan, Trp 21, which is expected to lie within alpha-helix 1 based on comparison with the X-ray crystal structures of the pi- and mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme A1-1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequency-domain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substrate-free GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9-16% of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the "H-site" is occupied.

Human Mu-class glutathione S-transferases present in liver, skeletal muscle and testicular tissue

The major human Mu-class glutathione S-transferases (GST) have been purified to allow comparisons of their catalytic, physicochemical and immunochemical properties. GST isoenzymes, purified from hepatic, testicular and skeletal muscle tissue were found to comprise three distinct subunits (M1, M2 and M3) which may combine to form both homodimeric and heterodimeric proteins. Two distinct subunits, M1a and M1b, which represent allelic charge variants have been isolated but no polymorphic forms encoded at the GST M2 and M3 loci have been observed. Three GST isoenzymes (M1a-1a, M1a-1b and M1b-1b) have been purified from a single liver specimen. In addition, GST M1a-2, M1b-2, M2-2 and M2-3 have been isolated from muscle, whilst the M3-3 homodimer has been purified from human testis. The homodimeric enzymes GST M1a-1a, M1b-1b, M2-2 and M3-3 have p I values of 6.1, 5.5, 5.3 and 5.0, whilst SDS-PAGE indicated that M1a, M1, M2 and M3 have molecular masses of 26.7, 26.6, 26.0 and 26.3 kDa, respectively. The M1, M2 and M3 subunits isolated from either liver, skeletal muscle or testis, are catalytically distinct. Both M1-type subunits (M1a and M1b) possess a high activity for trans-4-phenyl-3-buten-2-one, whereas, the skeletal muscle subunit M2 has a high activity towards 1,2-dichloro-4-nitrobenzene. By contrast, the testicular GST subunit M3 has no detectable activity towards either of these substrates. However, all three Mu-class subunits are active towards the compounds 4-hydroxynonenal and 4-hydroxydecinal, possible endogenous substrates which are produced by lipid peroxidation. The human Mu-class subunits can be distinguished immunochemically; antisera raised against the testicular GST M3-3 showed no reactivity towards either the M1 or M2 subunits. The M3 subunit has a blocked N-terminus but automated amino-acid sequencing of a CNBr-derived peptide allowed 14 residues of the M3 subunit to be identified. These data indicated that testicular GST M3-3 is likely to correspond to the brain/testis Mu-class GST cDNA described by Campbell et al. (Campbell E., Takahashi Y., Abramovitz M., Peretz M., & Listowsky I. (1990) J. Biol. Chem. 265, 9188–9193).

Stereoselectivity of human liver and intestinal cytosolic fractions as well as purified human glutathione S-transferase isoenzymes towards 2-bromoisovalerylurea enantiomers

Glutathione (GSH) conjugation of 2-bromoisovalerylurea (BIU) enantiomers is stereoselective in humans in vivo. Administration of racemic BIU results in a higher plasma elimination and urinary excretion of R-BIU and its mercapturate, respectively, than of S-BIU and its mercapturate. In order to relate the in vivo BIU pharmacokinetics to the activity of glutathioe S-transferase (GST) isoenzymes, the GSH conjugation of BIU enantiomers was studied with human liver and intestinal cytosolic fractions as well as purified human class alpha (GSTA1-1, GSTA2-2), mu (GSTM1a-1a) and pi (GSTP1-1) GST isoenzymes. Stereoselective GSH conjugation of BIU enantiomers was observed for most human liver and intestinal cytosolic fraction. In general, the cytosolic fractions preferentially conjugated S-BIU. Stereoselective preference for GSH conjugation of S-BIU was also observed for GSTA2-2 and GSTM1a-1a, whereas GSTA1-1 was not selective for either of the BIU enantiomers. GSTP1-1 did not catalyse conjugation of R- and S-BIU. Quantification of the GST isoenzymes in the liver cytosolic fractions showed that the stereoselectivity towards S-BIU was related to the profile and amount of GST subunits in the cytosolic fractions. The discrepancy in stereoselectivity between the BIU pharmacokinetics in vivo and the GSH conjugation of BIU enantiomers in vitro is discussed. In addition, since in contrast to human GSTM1a-1a, rat class Mu isoenzymes prefer R-BIU, the present results indicate that related isoenzymes in different species may have a different stereoselectivity.

Kinetic mechanism of human erythrocyte acidic isoenzyme ϱ

The kinetic mechanism has been determined for human glutathione S-transferase ϱ (rho), an isoenzyme related to the human π (pi) isoenzyme. The kinetic mechanism was investigated by both non-linear regression studies and the analysis of primary and secondary plots, utilizing initial rate and product inhibition data. It was concluded that human isoenzyme ϱ obeys a random sequential Bi-Bi rapid equilibrium mechanism with the formation of an enzyme-substrate-product (enzyme-CDNB-conjugate) dead-end complex. The values of K CDNB, K GSH and K conjugate were 0.70 ± 0.11, 0.12 ± 0.02 and 0.016 ± 0.004 mM, respectively. Comparison of the kinetic mechanism and kinetic parameters obtained for glutathione S-transferase isoenzyme ϱ with other class π isoenzymes showed similarities at the primary kinetic level.

Chromosomal Mapping of the Human Mu Class Glutathione S-Transferases to 1p13

The chromosomal localization of the human Mu class glutathione S-transferase (GST) genes has been complicated by two factors; the total number of genes is unknown and there is a polymorphism that results from the presence or absence of the GSTM1 gene. Three human Mu class glutathione S-transferase isoenzymes, GSTM1, GSTM2, and GSTM3, have been characterized previously, and we have recently cloned and characterized GSTM4, another member of this class. Here we report that a probe derived from GSTM4 cross-hybridizes with the other three known human Mu class GST genes. In situ hybridization with the GSTM4 probe localized a major region of hybridization on chromosome band 1p13. Although there is a region of very weak hybridization on chromosome 6, these data indicate that the human Mu class gene family is largely clustered and not dispersed on different chromosomes. The identical hybridization patterns in individuals with or without the GSTM1 gene suggest that this locus is a component of the Mu class GST gene cluster.

Glutathione Transferase Catalyzed Conjugation of Diol Epoxides Derived from Polycyclic Aromatic Hydrocarbons with Glutathione

Abstract Human glutathione transferase isoenzymes (GST) Al-1, Ml-1 and Pl-1 have been incubated with each of the four stereoisomers of trans-3,4-dihydroxy 1,2-epoxy l,2,3,4-tetrahydrobenzo[c]phenanthrene [(+)- and anti- and (+)-and (-)-syn-BPhDE] and glutathione (GSH). The results demonstrate that all BPhDE isomers were substrates for all the GST isoenzymes studied. However, a marked variation in catalytic efficiencies (> 10-fold) was noted. The enantiomers with 1R,2S-absolute configuration [(+)-syn- and (-)-anti-BPhDE] were in general better substrates than the corresponding 1S,2R-epoxides. Overall, GSTA1-1 was the most active isoenzyme. In preliminary experiments, a number of racemic bay- and fjord-region diol epoxides (derived from chrysene, benzo[c]- and [g]chrysene, dibenzo[a,h]anthracene and picene) have been incubated with GSTP1-1 and the formation of GSH-conjugates estimated. The results show that the syn-diastereomers in general were better substrates than the anti diastereomers. Furthermore, the enantiomers with R,S-epoxide absolute configuration were preferentially conjugated, thus demonstrating a high degree of selectivity.

Modulation of Detoxifying Potential and Differential Expression of Glutathione Transferase Isoenzyme in the Bacterial ada Gene-transfected Murine Hepatoma Cell

Modulation of Detoxifying Potential and Differential Expression of Glutathione Transferase Isoenzyme in the Bacterial ada Gene-transfected Murine Hepatoma Cell

Molecular cloning and heterologous expression of an alternatively spliced human Mu class glutathione S-transferase transcript.

Two cDNA clones encoding a new Mu class glutathione S-transferase (GST) have been isolated from a human testis cDNA library. Both clones are incomplete and appear to result from alternative splicing. One clone is missing the sequence encoding exon 4 and the other is missing exon 8. The complete sequence of the previously undescribed isoenzyme can be deduced from the two cDNA clones. This is the first report of alternative splicing in a GST transcript and may represent either a novel form of regulation in this multigene family or illegitimate transcription and experimental alternative splicing as part of the evolutionary process. By combining components from each clone a complete cDNA has been constructed and the encoded protein expressed in Escherichia coli. In general, the recombinant enzyme has relatively low activity when compared with all the previously described human Mu class GST isoenzymes.

Response to adjuvant chemotherapy in primary breast cancer: no correlation with expression of glutathione S-transferases.

Of 139 node-positive breast cancer patients treated with adjuvant chemotherapy, the pre-treatment levels of glutathione S-transferase (GST) classes alpha, mu and pi, were determined by immuno-quantification on Western blots in cytosols of the primary tumours. Their expression was studied with respect to cytosolic oestrogen-receptor, progesterone-receptor and cathepsin D levels, and to the length of disease-free survival. GST class pi was negatively correlated with oestrogen receptor and progesterone receptor, and positively correlated with cathepsin D. There was no correlation between GST isoenzymes and the length of disease-free survival. These data suggest that glutathione S-transferases are not useful as markers to predict the response to adjuvant chemotherapy in human breast cancer.

In vitro and in vivo reversible and irreversible inhibition of rat glutathione S-transferase isoenzymes by caffeic acid and its 2- S-glutathionyl conjugate

The reversible and irreversible inhibition of glutathione S-transferases (GST) by caffeic acid [3-(3,4-dihydroxyphenyl)-2-propenoic acid] was studied in vitro using purified rat isoenzymes, and in vivo in male Wistar (WU) rats. The concentrations of caffeic acid that inhibited reversibly 50% of the activity of different GST isoenzymes towards 1-chloro-2,4-dinitrobenzene (CDNB) (I 50 values) were 58 (GST 4-4), 360 (GST 3-3) and 470 μ m (GST 7-7), and higher than 640 μ m for GST isoenzymes of the α class (GST 1-1 and 2-2). The major glutathione conjugate of caffeic acid, 2- S-glutathionylcaffeic acid (2-GSCA), was a much more potent reversible inhibitor of GST, with I 50 values of 7.1 (GST 3-3), 13 (GST 1-1), 26 (GST 4-4), 36 (GST 7-7) and more than 125 μ m (GST 2-2). On the other hand, caffeic acid was a much more efficient irreversible inhibitor of GST than 2-GSCA. In this respect, GST 7-7 was by far the most sensitive enzyme. The remaining activity towards CDNB (expressed as percentage of control) after incubating 1.25 μ m-GST with 100 μ m-caffeic acid for 6 hr at 37°C was 34 (GST 2-2), 24 (GST 1-1), 23 (GST 4-4), 10 (GST 3-3) and 5% (GST 7-7). Almost no irreversible inhibition of GST 1-1 and 3-3 occurred during incubation with 2-GSCA. Incubation of caffeic acid with liver microsomes from dexamethasone-induced rats catalysed the oxidation of caffeic acid about 18 times more effectively as compared with the spontaneous oxidation, as determined by the formation of GSH conjugates from caffeic acid. In vivo, the effect of single oral doses of caffeic acid (50–500 mg/kg body weight) on the cytosolic GST activity towards CDNB was studied 18 hr after dosing in the liver, kidney and intestinal mucosa. A marginal but significant linear relationship was found between the amount of caffeic acid dosed and the irreversible inhibition of GST activity in the liver, with a maximum of about 14% inhibition in the highest dose group. This inhibition coincided with a small decrease in the μ-class GST subunits, which was only significant for GST subunit 4.

Glutathione analogue sorbents selectively bind glutathione S-transferase isoenzymes.

Novel affinity sorbents for glutathione S-transferases (GSTs) were created by binding glutathione (GSH) analogues to Sepharose 6B. The GSH molecule was modified at the glycine moiety and at the group attached to the sulphur of cysteine. When tested by affinity chromatography in a flow-through microplate format, several of these sorbents selectively bound GST isoenzymes. gamma E-C(Hx)-phi G (glutathione with a hexyl moiety bound to cysteine and phenylglycine substituted for glycine) specifically bound rat GST 7-7, the Pi-class isoenzyme, from liver, kidney and small intestine. gamma E-C(Bz)-beta A (benzyl bound to cysteine and beta-alanine substituted for glycine) was highly selective for rat subunits 3 and 4, which are Mu-class isoenzymes. By allowing purification of the isoenzymes under mild conditions that preserve activity, the novel sorbents should be useful in characterizing the biological roles of GSTs in both normal animal and cancer tissues.

Glutathione S-transferase isoenzymes in rat brain neurons and glia

The glutathione S-transferases (GSTs) constitute a family of cytosolic isoenzymes and a structurally unrelated microsomal enzyme that is involved in the detoxication of electrophilic xenobiotics. These enzymes also participate in the intracellular binding and transport of a broad range of lipophilic compounds including bilirubin, and hormones such as the glucocorticoids and thyroid hormones. The present investigation demonstrates that GSTs are present in neurons of the brainstem, forebrain, and cerebellum. An isoenzyme-specific distribution of GSTs was found in cytoplasm, nuclei, and nucleoli. The regional and cellular distribution of cytosolic GSTs in the brain was studied by immunohistochemistry, spectrophotometric enzyme assay, and reverse-phase HPLC. Polyclonal antibody against microsomal GST was strongly reactive with Purkinje cells throughout the cerebellar cortex, and with neurons in the brainstem and hippocampus. Nuclei of Purkinje cells and of neurons in the brainstem, hippocampus, and cerebral cortex were immunopositive for alpha-class GST 1-1 (YaYa), whereas alpha-class GST 2-2 (YcYc) antibody was consistently immunoreactive with the nucleolus, but not with the nucleus or soma. All alpha-class GST antibodies studied were reactive, to various degrees, with astrocytes and choroid plexus; however, ependymal cells of the subventricular zones were immunonegative. alpha-class GST 8-8 (YkYk) immunoreactivity was specifically localized to endothelial cells and/or astrocytic end feet associated with blood vessels. Reverse-phase HPLC indicated that there were also substantial regional differences in the pattern of alpha-, mu-, and pi-class GST subunit expression. For example, the thalamus/hypothalamus had the highest GST activity and greatest concentration of total GST protein and mu-class GST subunit 6 (Yb3), whereas the brainstem had the greatest concentration of pi-class GST subunit (Yp). This regional variation in GST expression may be reflective of regional differences in cell populations. In cerebellar cortex, the concentration of mu-class GST subunit 4 (Yb2) was greatest in the flocculus and lowest in the vermis. This is of clinical interest because the pattern of expression of mu-class GST subunit 4 (Yb2) in the cerebellum coincides with the known regional susceptibility of this structure to degeneration after exposure to toxic or metabolic insults. The vermis is most susceptible to these insults, whereas the lateral lobes and flocculus are most resistant.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental aspects of Bufo bufo embryo glutathione transferases

The expression of glutathione transferase isoenzymes has been studied during the development of Bufo bufo embryo. By analysing the GSH-affinity purified materials in terms of substrate specificities, SDS-PAGE pattern, HPLC elution profile, we conclude that, up to stage 22, no significant changes in the expression of glutathione transferases isoenzymes occurred during Bufo bufo embryo development. At stage 25 the distribution of glutathione transferases was found to be slightly different from those of all other foregoing stages. A marked decrease of embryonic glutathione transferases subunits with a parallel appearance of new structurally and immunologically different subunits was noted in toad liver and kidney. Toad ovary continued to express embryonic glutathione transferase subunits.

Effects of hyperbilirubinaemia on glutathione S-transferase isoenzymes in cerebellar cortex of the Gunn rat.

The glutathione S-transferases (GSTs) are a family of isoenzymes involved in the detoxication of a variety of electrophilic xenobiotics. The present investigation demonstrates that GST activity and the concentration of cytosolic GSTs in cerebellar cortex of Gunn rats were increased in hyperbilirubinaemic animals compared with non-jaundiced controls. Age-dependent and region-specific increases in GST isoenzymes were seen in three regions of the cerebellar cortex of jaundiced Gunn rats, whereas GST concentrations were not altered in the brainstem, thalamus/hypothalamus, cortex or liver. Cytosolic GST activity was increased 1.3-fold in the flocculus and lateral hemispheres of 20-day-old and 1.7-fold in the flocculus, lateral hemispheres and vermis of 60-day-old jaundiced (jj; homozygous) Gunn rats compared with non-jaundiced (Jj; heterozygous) Gunn rats. H.p.l.c. was used to determine the GST subunit protein concentrations in cytosolic fractions isolated from liver and brain regions of jaundiced and non-jaundiced animals. In all regions of the cerebellum from 20-day-old animals, the levels of Alpha-class GST subunits 2 (Yc1; 3.0-fold) and 8 (Yk; 2.0-fold) were increased in jaundiced rats. In 60-day-old animals, the concentrations of Alpha-class GST subunits 2 (Yc1; 5.0-fold) and 8 (Yk; 3.0-fold), Mu-class subunit 11 (Yo; 2.5-fold) and Pi-class subunit 7 (Yp; 2.0-fold) were increased in all regions of cerebellar cortex of jaundiced animals. In cerebellum of 10-, 20- and 60-day-old non-jaundiced and jaundiced Gunn rats, the flocculus had the highest concentration of Mu-class GST subunit 4 (Yb2) and vermis the lowest; hyperbilirubinaemia increased the concentration of subunit 4 (Yb2; 3- to 5-fold) in the flocculus and lateral hemispheres, but not the vermis, of 20- and 60-day-old rats. Intraperitoneal injection of sulphadimethoxine, a long-acting sulphonamide which displaces bilirubin from its albumin-binding sites and increases the bilirubin levels in tissues, further increased the already elevated concentrations of GST subunits in the lateral regions of cerebellar cortex of hyperbilirubinaemic rats. For example, the concentration of subunit 4 (Yb2) was increased 2.2-fold (compared with non-jaundiced controls) in Gunn rats injected with saline and 7.4-fold in rats injected with 100 mg of sulphadimethoxine/kg body weight. In contrast, GSTs in the vermis of jaundiced animals were not affected by sulphadimethoxine injection. Sulphadimethoxine had no effect on GST concentrations in lateral regions and vermis of heterozygous (Jj) Gunn rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Activity of Mouse Liver Glutathione S-Transferases toward trans,trans-Muconaldehyde and trans-4-Hydroxy-2-nonenal

This study investigated the catalytic activities of hepatic glutathione S-transferase (GST) isoenzymes isolated from CD-1 mice toward two activated alkenals of toxicological relevance: trans,trans-muconaldehyde (MA), a putative myelotoxic metabolite of benzene, and trans-4-hydroxy-2-nonenal (HNE), a highly reactive lipid peroxidation product. The activity toward 1-chloro-2,4-dinitrobenzene (CDNB) was also determined. Four isoenzymes with p I values of 9.8, 8.7, 6.4, and 5.7 were each isolated from male and female mice. The isoenzymes with p I values of 8.7 and 6.4 are π and μ class GSTs, respectively, whereas the p I 9.8 and 5.7 GSTs are both α class isoenzymes. CDNB activity was greatest in the π (p I 8.7) isoenzyme of both sexes. In addition, the CDNB activity of the π (p I 8.7) isoenzyme from males was markedly greater than the corresponding GST from female mouse liver. In contrast to CDNB, both MA and HNE were better substrates for the acidic α (p I 5.7) and μ (p I 6.4) GSTs, whereas minimal activity toward either alkenal was detected in the π (p I 8.7) and α (p I 9.8) isoenzymes. Maximum activity toward MA and HNE was exhibited by the α (p I 5.7) isoenzyme of both sexes. The level of HNE activity observed with the α (p I 5.7) isoenzyme was five- to sixfold greater than that reported previously for any mouse GST isoenzyme. Moreover, the specific activities of the female α (p I 5.7) isoenzyme toward both HNE and MA were markedly greater than those of the corresponding isoenzyme from males. A similar gender-specific difference was noted in the activity of the μ (p I 6.4) isoenzyme toward HNE, but not toward MA. These results show that both MA and HNE are substrates for the α (p I 5.7) and μ (p I 6.4) GSTs of murine liver, with maximum activity toward both activated alkenals exhibited by the α (p I 5.7) isozyme. In addition, evidence is presented that demonstrates a female-dominant sex difference in the activity of the α (p I 5.7) isoenzyme toward MA and HNE, which contrasts sharply with the male-dominant activity of π class GSTs toward CDNB. These results are consistent with the hypothesis that α and μ class GSTs are critical detoxication enzymes in female mouse liver, whereas π-class GST isozymes predominate in the liver of male mice.

Isoenzyme selective irreversible inhibition of rat and human glutathione S-transferases by ethacrynic acid and two brominated derivatives

In the present study it has been shown that ethacrynic acid can inhibit glutathione S-transferase (GST) of the pi-class irreversibly. [ 14C]Ethacrynic acid, 0.8 nmol/nmol human P1-1 and 0.8 nmol/nmol rat GST 7-7 could be incorporated, resulting in 65–93% inhibition of the activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Isoenzymes of the alpha- and mu-class also bound [ 14C]ethacrynic acid, however without loss of catalytic activity. Incorporation ranged from 0.3 to 0.6 and 0.2 nmol/nmol enzyme for the mu- and alpha-class GST isoenzymes, respectively. For all isoenzymes, incorporation of [ 14C]ethacrynic acid could be prevented by preincubation with tetrachloro-1,4-benzoquinone, suggesting, that a cysteine residue is the target site. Protection of GST P1-1 against inhibition by ethacrynic acid by the substrate analog S-hexylglutathione, indicates an active site-directed modification. The monobromo and dibromo dihydro derivatives of ethacrynic acid were synthesized in an effort to produce more reactive compounds. The monobromo derivative did not exhibit enhanced irreversible inhibitory capacity. However, the dibromo dihydro derivative inhibited both human and rat GST isoenzymes of the pi-class very efficiently, resulting in 90–96% inhibition of the activity towards CDNB. Interestingly, this compound is also a powerful irreversible inhibitor of the mu-class GST isoenzymes, resulting in 52–70% inhibition. The two bromine atoms only marginally affect the strong (reversible) competitive inhibitory capacity of ethacrynic acid, with ic 50 (μM) of 0.4–0.6 and 4.6–10 for the mu- and pi-class GST isoenzymes, respectively.

Distribution and activity of glutathione-S-transferase in normal human lenses and in cataractous human epithelia.

The distribution of glutathione-S-transferase (GST) activity was determined in frozen normal human lenses. The highest activity of GST was found in the peripheral and equatorial regions, whereas the lowest activity was found in the nucleus. Western blot showed that both mu and pi isoenzymes of GST were present in human lenses. This result is similar to that found in rat lenses. In addition, GST activity was analyzed in 50 lens epithelia which were obtained during cataract surgery. Twenty-seven lens epithelia showed no activity. Statistically significant association was found between cortical and mixed cortical--nuclear cataract and loss of GST activity. No association was found between pure nuclear cataract and loss of epithelial GST activity.