Atrazine Resistance in a Velvetleaf (Abutilon theophrasti) Biotype Due to Enhanced Glutathione S-Transferase Activity

We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or “wild-type” velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K_m values for glutathione, atrazine, or 1-chloro-2,4-dinitrobenzene. However, the V_max for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V_max for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation.     

Partial characterization of glutathione S-transferases from wheat (Triticum spp.) and purification of a safener-induced glutathione S-transferase from Triticum tauschii.

Hexaploid wheat (Triticum aestivum L.) has very low constitutive glutathione S-transferase (GST) activity when assayed with the chloroacetamide herbicide dimethenamid as a substrate, which may account for its low tolerance to dimethenamid in the field. Treatment of seeds with the herbicide safener fluxofenim increased the total GST activity extracted from T. aestivum shoots 9-fold when assayed with dimethenamid as a substrate, but had no effect on glutathione levels. Total GST activity in crude protein extracts from T. aestivum, Triticum durum, and Triticum tauschii was separated into several component GST activities by anion-exchange fast-protein liquid chromatography. These activities (isozymes) differed with respect to their activities toward dimethenamid or 1-chloro-2,4-dinitrobenzene as substrates and in their levels of induction by safener treatment. A safener-induced GST isozyme was subsequently purified by anion-exchange and affinity chromatography from etiolated shoots of the diploid wheat species T. tauschii (a progenitor of hexaploid wheat) treated with the herbicide safener cloquintocet-mexyl. The isozyme bound to a dimethenamid-affinity column and had a subunit molecular mass of 26 kD based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme (designated GST TSI-1) was recognized by an antiserum raised against a mixture of maize (Zea mays) GSTs. Amino acid sequences obtained from protease-digested GST TSI-1 had significant homology with the safener-inducible maize GST V and two auxin-regulated tobacco (Nicotiana tabacum) GST isozymes.     

Glutathione S-transferases in elasmobranch liver. Molecular heterogeneity, catalytic and binding properties, and purification

In order to gain insight into the phylogeny and physiological significance of organic-anion-binding proteins in the liver, the hepatic glutathione S-transferases of rat and a typical elasmobranch, the thorny-back shark (Platyrhinoides triseriata), were compared with respect to both glutathione S-transferase activites and organic-anion-binding properties. On gel filtration (Sephadex G-75, Superfine grade) of rat cytosol, the elution volumes of enzyme activities with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates were identical (rat Y-fractions; M_r 45000). In contrast, two peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to M_r 52000 (PLAT Y₁) and M_r 45000 (PLAT Y₂) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y₂ had enzyme activity with p-nitrobenzyl chloride. Enzyme kinetic studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y₁- and PLAT Y₂-fractions. The two forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that we found to be physiological in P. triseriata, PLAT Y₂ accounted for approx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y₁ and PLAT Y₂ fractions had much lower affinities for sulphobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y₁ and PLAT Y₂ for Rose Bengal and 1-anilino-8-naphthalenesulphonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulphobromophthalein and Rose Bengal were non-competitive inhibitors of glutathione S-transferase activities when 1-chloro-2,4-dinitrobenzene was used as substrate for both PLAT Y₁ and PLAT Y₂. The major glutathione S-transferase from the PLAT Y₂ fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of two identical subunits with pI7.7. The highly enriched Y₂-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulphonate.     

The hypothalamic–hypophyseal–gonadal regulation of hepatic glutathione S-transferases in the rat

Hepatic glutathione S-transferase activities were determined with the substrates 1,2-dichloro-4-nitrobenzene and 1-chloro-2,4-dinitrobenzene. Sexual differentiation of glutathione S-transferase activities is not evident during the prepubertal period, but glutathione conjugation with 1,2-dichloro-4-nitrobenzene is 2–3-fold greater in adult males than in females. Glutathione conjugation with 1-chloro-2,4-dinitrobenzene is slightly higher in adult males than adult females. No change in activity was observed after postpubertal gonadectomy of males or females. Neonatal castration of males results in a significant decrease in glutathione conjugation with 1,2-dichloro-4-nitrobenzene. Hypophysectomy, or hypophysectomy followed by gonadectomy did result in significantly higher glutathione S-transferase activities in both sexes. These increases can be reversed by implanting an adult male or female pituitary or four prepubertal pituitaries under the kidney capsule. Postpubertal sexual differentiation of glutathione S-transferase activities is neither dependent on pituitary sexual differentiation nor pituitary maturation. Prolactin concentrations are inversely related to glutathione S-transferase activities in hypophysectomized rats with or without ectopic pituitaries. Somatotropin exogenously administered to hypophysectomized rats results in decreased glutathione S-transferase activities, whereas prolactin has no effect. Adult male rats treated neonatally with monosodium l-glutamate to induce arcuate nucleus lesions of the hypothalamus have decreased glutathione S-transferase activities towards 1,2-dichloro-4-nitrobenzene and decreased somatotropin concentrations. Our experiments suggests that sexual differentiation of hepatic glutathione S-transferase is a result of a hypothalamic inhibiting factor in the male (absent in the female). This postpubertally expressed inhibiting factor acts on the pituitary to prevent secretion of a pituitary inhibiting factor (autonomously secreted by the female), resulting in higher glutathione S-transferase activities in the adult male than the adult female.     

Comparison of glutathione S-transferases of Zea mays responsible for herbicide detoxification in plants and suspension-cultured cells

The metabolism of the s-triazine herbicide atrazine has been compared in Zea mays seedlings and cell suspension cultures. The rapid detoxification observed in the shoots of whole plants was not seen in the cultured cells. This difference in metabolism could be accounted for by the varying substrate specificities of the isoenzymes of glutathione S-transferase (EC 2.5.1.18) present in the plant and the cells. A single form of the enzyme isolated from leaf tissue conjugated both atrazine and the chloracetanilide herbicide metolachlor. However, the two isoenzymes present in suspension-cultured cells although active against metolachlor, showed no activity toward atrazine. Following purification, the major form of transferase present in the cells was physically similar to the enzyme isolated from leaf (M_r=55000). Both proteins were dimers of subunit M_r=26300, and with isoelectric points in the range pH 4.3-4.9. The minor form of the enzyme present in culture showed a greater specificity for metolachlor than the major species. In addition the overall activity and ratio of the two isoenzymes varied over the culture growth cycle. These findings illustrate the need for characterizing enzymes involved in herbicide detoxification in plant cell cultures.Abbreviations CDNB 1-chloro-2,4-dinitrobenzene - DEAE diethylaminoethyl - GSH glutathione (reduced) - GST glutathione S-transferase - HPLC high-pressure liquid chromatography - M_r molecular weight - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Different rates of metabolism of two chloroacetanilide herbicides in pioneer 3320 corn

The in vivo rates of uptake and detoxification of alachlor and metolachlor were determined using Pioneer corn 3320 seedlings. Equal amounts of the radiolabeled herbicides were applied to etiolated coleoptiles and, at various intervals after treatment, the unabsorbed radioactivity was removed and quantified. Analysis of 80% methanol extracts by reverse phase liquid chromatography showed no significant differences in the rate of uptake of metolachlor and alachlor. However, the rate of glutathione conjugation of alachlor in vivo was two- to threefold greater than the rate for metolachlor at 2 and 4 hours after herbicide application. Since the initial step in detoxification is conjugation of the chloroacetanilide to glutathione, the activities of the enzymes responsible for conjugation, the glutathione-S-transferases (GST) were also analyzed in vitro, using crude extracts and the purified GST enzymes. The specific activities of the extracts were consistent with the results in vivo. Using alachlor as a substrate, the specific activity for glutathione conjugation was almost threefold higher than that for metolachlor. Kinetic analysis of purified GST III indicates that the enzyme has a higher affinity for alachlor (K_mapp = 1.69 millimolar) than for metolachlor (K_mapp = 8.9 millimolar).     

A study of gender,strain and age differences in mouse liver glutathione-S-transferase

The hepatic cytosolic glutathione S-transferase (GST) activity in four strains of the mouse and one strain of the rat was studied with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethachrynic acid (ETHA), cumene hydroperoxide (CU) and atrazine as the in vitro substrates. In the mouse, significant gender, strain and age-related differences in the GST activity towards CDNB and atrazine were found between adolescent and sexually mature males and females of the CD-1, C57BL/6, DBA/2 and Swiss-Webster strains, and the differences were larger with atrazine as the substrate. With DCNB and CU a similar tendency was observed, however not significant for all strains. The GST activity towards ETHA was also gender and strain specific, but revealed no age-related differences. The herbicide atrazine seems to be a useful substrate in the study of strain and age-related differences in the mouse GST class Pi.     

Characterisation of glutathione transferases and glutathione peroxidases in pea (Pisum sativum)

Glutathione peroxidases (GPOXs) and glutathione transferases, also termed glutathione S-transferases (GST, EC 2.5.1.18), with activities toward a range of xenobiotic substrates including herbicides, have been characterized in etiolated pea (Pisum sativum L. cv. Feltham's First) seedlings. Crude extracts showed high activity toward a range of GST substrates including 1-chloro-2,4-dinitrobenzene (GSTC activity) and the herbicide fluorodifen (GSTF) but low activities toward chloroacetanilides and atrazine. Treatment of the pea seedlings with the herbicide safener dichlormid selectively increased the activity of GSTC and the GST which detoxified atrazine. This induction was restricted to the roots and was not observed with any of the other GST or GPOX activities. In contrast, treatment with CuCl₂ increased GPOX activity in the root but had no effect on any GST activity, while treatment of epicotyls with elicitors of the phytoalexin response increased GST activity toward ethacrynic acid, but had no effect on other GST or GPOX activities. The major enzymes with GSTC, GSTF and GPOX activities were purified from pea epicotyls 3609-fold, 1431-fold and 1554-fold, respectively. During purification by hydrophobic interaction chromatography and affinity chromatography using S-hexyl-glutathione as ligand all three activities co-eluted but could be partially resolved by anion exchange chromatography and gel filtration chromatography. Both GSTC and GPOX had a molecular mass of 48 kDa and their activities were associated with a similar 27.5-kDa subunit but distinct 29-kDa subunits. GSTF could be resolved into two isoenzymes with molecular masses of 49.5 and 54 kDa. GSTF activity was associated with a unique 30-kDa subunit in addition to 27.5- and 29-kDa peptides, suggesting that the two isoenzymes were composed of differing subunits. These results demonstrate that peas contain multiple GST isoenzymes some of which have GPOX activity and that the various activities are differentially responsive to biotic and abiotic stress.     

Glutathione S-Transferase Isoenzymes from Streptomyces griseus

An inducible, cytosolic glutathione S-transferase (GST) was purified from Streptomyces griseus. GST isoenzymes with pI values of 6.8 and 7.9 used standard GST substrates including 1-chloro-2,4-dinitrobenzene. GST had subunit and native M_rs of 24 and 48, respectively, and the N-terminal sequence SMILXYWDIIRGLPAH.     

The isolation of a fetal rat liver glutathione S-tranferase isoenzyme with high glutathione peroxidase activity

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Y_c (M_r 28 000) and a subunit (M_r 25 500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit ‘Y_fetus’. The enzyme which we term glutathione S-transferase Y_cY_fetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.     

Structure and Expression of a Dhurrinase (β-Glucosidase) from Sorghum

Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression.     

Identification of a new glutathione S-transferase from rat liver cytosol

A new glutathione $\text{S}$-transferase has been purified to homogeneity from 105,000 × g supernatant of Sprague-Dawley rat liver homogenates. The purified enzyme exhibited specific activities of approximately 1.8, and 0.12 μmoles. min^?1. mg^?1 toward 1-chloro 2,4-dinitrobenzene and cumene hydroperoxide respectively. The SDS gel electrophoresis data on subunit composition revealed that the new transferase is composed of two subunits with an identical M_r of 24,400 (Y_α Family). Our $\text{in}\text{vitro}$ translation experiments with rat liver poly(A) RNAs and substrate specificity data suggest that this subunit is different from the previously reported Ya, Yb and Yc subunits of rat liver glutathione $\text{S}$-transferases. Comparatively, the new isozyme showed significant activity toward 1,2 epoxy-3-(P-nitrophenoxy)-propane, ethacrynic acid and P-nitrophenyl acetate, 0.4, 0.34 and 0.18 μ moles. min^?1. mg^?1 respectively.     

Selective inhibition of glutathione S-transferases by 17 beta-estradiol disulfate.

Enzyme activity of homogeneous glutathione S-transferases A, B, and C with reduced glutathione and 1-chloro-2,4-dinitrobenzene was inhibited in varying degrees by 50 μm concentrations of monosulfate and disulfate derivatives of several steroids. In contrast, transferase AA activity was not affected. Of the inhibitors tested, estradiol-3,17-disulfate and estradiol-3-sulfate were the most inhibitory, followed by pregnenolone sulfate, estradiol-17-sulfate, dehydroisoandrosterone sulfate, and cortisol sulfate. Transferases A and C were most affected, especially by estradiol disulfate and estradiol-3-sulfate, which exhibited essentially complete inhibition at a concentration of μm. Double reciprocal plots of estradiol disulfate inhibition with respect to 1-chloro-2,4-dinitrobenzene concentration showed uncompetitive inhibition with transferases A and C and noncompetitive inhibition with transferase B (ligandin). With reduced glutathione as the variable substrate, transferases A and C exhibited noncompetitive inhibition kinetics, while transferase B showed partial noncompetitive kinetics.     

A radioenzymatic ultramicro method applicable to the measurement of a wide range of metabolites

A procedure for the rapid identification of glutathione S-transferase isozymes from rat liver in polyacrylamide gels is described. The isozymes are separated by electrofocusing and then identified by bathing the gels in a solution containing substrates and scanning the gels at the appropriate wavelength for the appearance of product. Increase in absorbance as a function of time delineates areas containing enzyme from artifacts within the gel. This technique should be useful for the identification of isozymes of glutathione S-transferase in other tissues and also other species. Also, the technique provides for rapid confirmation of homogeneity of the isozymes of glutathione S-transferase.     

A novel glutathione S-transferase isozyme similar to GST 8-8 of rat and mGSTA4-4 (GST 5.7) of mouse is selectively expressed in human tissues

A mouse glutathione S-transferase (GST) isozyme designated as GST 5.7 or mGSTA4-4 belongs to a distinct subclass of the α-class isozymes of GST. It is characterized by kinetic properties intermediate between the α- and π-classes of GSTs. We have recently cloned and expressed this isozyme (rec-mGSTA4-4) in E. coli and have reported its complete primary sequence (Zimniak, P. et al. (1992) FEBS Lett., 313, 173–176). Using antibodies raised against the homogenous rec-mGSTA4-4 expressed in E. coli, we now demonstrate that an ortholog of this isozyme was selectively expressed in various human tissues. The human ortholog of mGST A4-4 purified from liver had a pI value of 5.8 and constituted approx. 1.7% of total GST protein of human liver. Similar to other α-class GSTs, the N-terminus of this isozyme (GST 5.8) was also blocked. CNBr digestion of the enzyme yielded two major fragments with M_r values of 12 kDa and 6 kDa. The sequences of these two fragments showed identities in 16 out of 20 residues and 17 out of 20 residues with the corresponding sequences of its mouse ortholog (mGSTA4-4), and showed significant homologies with the rat and chicken orthologs, GST 8-8 and GST CL3. Human liver GST 5.8 showed more than an order of magnitude higher activity towards t-4-hydroxy-2-nonenal as compared to 1-chloro-2,4-dinitrobenzene. This isozyme also expressed glutathione-peroxidase activity towards fatty acid, as well as phospholipid hydroperoxidase suggesting its role in protection mechanisms against the toxicants generated during lipid peroxidation. Western blot analysis of human tissues revealed that this GST isozyme was selectively expressed in human liver, pancreas, heart, brain and bladder tissues, but absent in lung, skeletal muscle, spleen and colon.     

Purification and partial characterization of the glutathione S-transferase of rat erythrocytes

The single glutathione S-transferase (EC 2.5.1.18) present in rat erythrocytes was purified to apparent homogeneity by affinity chromatography on glutathione-Sepharose and hydroxyapatite chromatography. Approx. 1.86 mg enzyme is found in 100 ml packed erythrocytes and accounts for about 0.01% of total soluble protein. The native enzyme (M_r 48 000) displays a pI of 5.9 and appears to possess a homodimeric structure with a subunit of M_r 23 500. Enzyme activities with ethacrynic acid and cumene hydroperoxide were 24 and 3%, respectively, of that with 1-chloro-2,4-dinitrobenzene. The K_m values for 1-chloro-2,4-dinitrobenzene and glutathione were 1.0 and 0.142 mM, respectively. The concentrations of certain compounds required to produce 50% inhibition (I₅₀) were as follows: 12 μM bromosulphophthalein, 34 μM S-hexylglutathione, 339 μM oxidized glutathione and 1.5 mM cholate. Bromosulphophthalein was a noncompetitive inhibitor with respect to 1-chloro-2,4-dinitrobenzene (K_i = 8 μM) and glutathione (K_is = 4 μM; K_ii = 11.5 μM) while S-hexylglutathione was competitive with glutathione (K_i = 5 μM).     

Purification and characterization of a glutathione S-transferase from benoxacor-treated maize (Zea mays).

A glutathione S-transferase (GST) isozyme from maize (Zea mays Pioneer hybrid 3906) treated with the dichloroacetamide herbicide safener benoxacor (CGA-154281) was purified to homogeneity and partially characterized. The enzyme, assayed with metolachlor as a substrate, was purified approximately 200-fold by ammonium sulfate precipitation, anion-exchange chromatography on Mono Q resins, and affinity chromatography on S-hexylglutathione agarose from total GST activity present in etiolated shoots. The purified protein migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) as a single band with a molecular mass of 27 kD. Using nondenaturing PAGE, we determined that the native protein has a molecular mass of about 57 kD and that the protein exists as a dimer. Two-dimensional electrophoresis revealed only a single protein with an isoelectric point of 5.75 and molecular mass of 27 kD. These results further suggest that the protein exists as a homodimer of two identical 27-kD subunits. The enzyme was most active with substrates possessing a chloroacetamide structure. trans-Cinnamic acid and 1-chloro-2,4-dinitrobenzene were not effective substrates. Apparent Km values for the enzyme were 10.8 microM for the chloroacetamide metolachlor and 292 microM for glutathione. The enzyme was active from pH 6 to 9, with a pH optimum between 7.5 and 8. An apparently blocked amino terminus of the intact protein prevented direct amino acid sequencing. The enzyme was digested with trypsin, and the amino acid sequences of several peptide fragments were obtained. The sequence information for the isolated GST we have designated "GST IV" indicates that the enzyme is a unique maize GST but shares some homology with maize GSTs I and III.     

Cloning and characterization of a glutathione S-transferase induced by a herbicide safener in barley (Hordeum vulgare)

The effect of the herbicide safener mefenpyr-diethyl on glutathione S -transferase (GST, EC 2.5.1.18) activities of dark-grown barley ( Hordeum vulgare cv. Alexis) was examined. Mefenpyr-diethyl treatment increased the GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) and the herbicide fenoxaprop as substrates. Glutathione (GSH) peroxidase activity was markedly increased. GST subunits were analysed by high performance liquid chromatography (HPLC). The quantities of two major subunits were increased by the safener treatment, while the quantities of two other major subunits remained constant. A cDNA encoding the most abundant inducible GST (HvGST6) was cloned and expressed in E. coli . The purified enzyme exhibited a low activity with herbicides as substrates. By contrast, it exhibited a strong GSH peroxidase activity.     

Glutathione S-transferase activities in rat and mouse sperm and human semen

Glutathione $\text{S}$-transferase activity was found in sperm of the rat and $\text{DBA}\text{2J}$ and C57 $\text{BL}\text{6J}$ mice. In rat sperm activities with benzo(a)pyrene 4,5-oxide, styrene 7,8-oxide, and 1-chloro-2,4-dinitrobenzene were 0.88, 1.07, and 26.1 nmoles/min/mg protein, respectively. Δ⁵-3-Ketosteroid isomerase activity of rat sperm was 4.9 nmoles/min/mg protein. These specific glutathione $\text{S}$-transferase and Δ⁵-3-ketosteroid isomerase activities in sperm represent 0.4–4.1% of rat liver cytosol values. Human semen also contained significant glutathione $\text{S}$-transferase activity. It is postulated that these enzymes could function in the metabolism and detoxification of certain electrophilic xenobiotics, if present in sperm.