The major glutathione S-transferase in salmonid fish livers is homologous to the mammalian pi-class GST.

1. The hepatic glutathione S-transferase (GST) isoenzymes were isolated and characterized from salmon, sea trout and rainbow trout. 2. In all three species the predominant GST expressed comprised subunits of Mr 24,800. These subunits each co-migrated with the rat pi-class Yf polypeptide during SDS/polyacrylamide gel electrophoresis. 3. Western blotting experiments demonstrated immunochemical cross-reactivity between the major salmonid and the rat pi-class GSTs. 4. The salmon GST of subunit Mr 24,800 was digested with cyanogen bromide and the peptides, once purified by reverse-phase HPLC, were subjected to automated amino acid sequencing. 5. Over the region sequenced, the salmon GST possessed about 65% homology with the rat and human pi-class GST.     

Hepatic glutathione S-transferases in rainbow trout and their interaction with 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone

Glutathione S-transferase in the cytosol of rainbow trout liver was partially purified by affinity chromatography on a column with glutathione coupled to epoxy-activated Sepharose 6B, which retained 94% of the total activity. Chromatofocussing on a Polybuffer exchanger 118 column separated the glutathione S-transferase into six major cationic isoenzymes (K1-K6), and some minor fractions. SDS-polyacrylamide slab gel electrophoresis showed K1-K3 to be heterodimers with subunits of Mr 25,000 and 26,500, and K4-K6 to be homodimers with subunits of Mr 25,000. The glutathione S-transferase isoenzymes were partially characterized by different biochemical parameters. The hepatic rainbow trout glutathione S-transferases were inhibited by the organic water pollutants, 1,4-benzoquinone and 2,4-dichlorophenoxyacetic acid. The same kinetic inhibition patterns were observed with these inhibitors as for rat liver glutathione S-transferases. It is concluded that rainbow trout glutathione S-transferases can play a key role in the detoxication of organic micropollutants in the aquatic environment.     

Differential induction of class alpha glutathione S-transferases in mouse liver by the anticarcinogenic antioxidant butylated hydroxyanisole. Purification and characterization of glutathione S-transferase Ya1Ya1.

A novel cytosolic Alpha class glutathione S-transferase (GST) that is not normally expressed in mouse liver was found to be markedly induced (at least 20-fold) by the anti-carcinogenic compound butylated hydroxyanisole. This enzyme (designated GST Ya1 Ya1) did not bind to either the S-hexylglutathione-Sepharose or the glutathione-Sepharose affinity matrices, and purification was achieved by using bromosulphophthalein-glutathione-Sepharose. The purified isoenzyme, which comprises subunits of Mr 25,600, was characterized, and its catalytic, electrophoretic, immunochemical and structural properties are documented. GST Ya1 Ya1 was shown to be distinct from the Alpha class GST that is expressed in normal mouse liver and is composed of 25,800-Mr subunits; the Alpha class isoenzyme that is constitutively expressed in the liver is now designated GST Ya3 Ya3. Hepatic concentrations of GST Ya3 Ya3 were not significantly affected when mice were treated with butylated hydroxyanisole. Both Pi class GST (subunit Mr 24,800) and Mu class GST (subunit Mr 26,400) from female mouse liver were induced by dietary butylated hydroxyanisole. By contrast, hepatic concentrations of microsomal GST (subunit Mr 17,300) were unaffected.     

Purification and subunit-structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker

Five glutathione S-transferase (GST, EC 2.5.1.18) forms were purified from human liver by S-hexylglutathione affinity chromatography followed by chromatofocusing, and their subunit structures and immunological relationships to rat liver glutathione S-transferase forms were investigated. They were tentatively named GSTs I, II, III, IV and V in order of decreasing apparent isoelectric points (pI) on chromatofocusing. Their subunit molecular weights assessed on SDS-polyacrylamide gel electrophoresis were 27 (Mr X 10(-3)), 27, 27.7,27 and 26, respectively, (26, 26, 27, 26, and 24.5 on the assumption of rat GST subunit Ya, Yb and Yc as 25, 26.5 and 28, respectively), indicating that all forms are composed of two subunits identical in size. However, it was suggested by gel-isoelectric focusing in the presence of urea that GSTs I and IV are different homodimers, consisting of Y1 and Y4 subunits, respectively, which are of identical Mr but different pI, while GST II is a heterodimer composed of Y1 and Y4 subunits. This was confirmed by subunit recombination after guanidine hydrochloride treatment. GST III seemed to be identical with GST-mu with regard to Mr and pI. GST V was immunologically identical with the placental GST-pi. On double immunodiffusion or Western blotting using specific antibodies to rat glutathione S-transferases, GST I, II and IV were related to rat GST 1-1 (ligandin), GST III(mu) to rat GST 4-4 (D), and GST V (pi) to rat GST 7-7 (P), respectively. GST V (pi) was increased in hepatic tumors.     

The effects of selenium and copper deficiencies on glutathione S-transferase and glutathione peroxidase in rat liver.

Selenium (Se) deficiency in rats produced significant increases in the activity of hepatic glutathione S-transferase (GST) with 1-chloro-2,4-dinitrobenzene as substrate and in various GST isoenzymes when determined by radioimmunoassay. These changes is GST activity and concentration were associated with Se deficiency that was severe enough to provoke decreases of over 98% in hepatic Se-containing glutathione peroxidase activity (Se-GSHpx). However, decreases in hepatic Se-GSHpx of 60% induced by copper (Cu) deficiency had no effect on GST activity or concentration. Increased GST activity in Se deficiency has previously been postulated to be a compensatory response to loss of Se-GSHpx, since some GSTs have a non-Se-glutathione peroxidase (non-Se-GSHpx) activity. However, the GST isoenzymes determined in this study, GST Yb1Yb1, GST YcYc and GST YaYa, are known to have up to 30-fold differences in non-Se-GSHpx activity, but they were all significantly increased to a similar extent in the Se-deficient rats.     

Hepatic and branchial glutathione S-transferases of two fish species: Substrate specificity and biotransformation of microcystin-LR

Liver and gills of roach (Rutilus rutilus) and silver carp (Hypophthalmichthys molitrix) were examined for glutathione S-transferases (GSTs) contents and their substrate specificity and capacity to biotransform microcystin-LR (MC-LR). GSTs and other glutathione (GSH) affine proteins were purified using a GSH-agarose matrix and separated by anionic chromatography (AEC). Substrate specificities were determined photometrical for 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrobenzyl chloride (pNBC) and ethacrynic acid (ETHA). Biotransformation rate of MC-LR was determined by high performance liquid chromatography (HPLC). Roach exhibited different hepatic and branchial GST activities for used substrates (DNB, pNBC and DCNB) compared to silver carp but not for ethacrynic acid. It suggests that, both fish species have similar amount of pi and/or alpha class, which were the dominant GST classes in liver and gills. Gills of both fish species contained a higher number of GST isoenzymes, but with lower activities and ability of MC-LR biotransformation than livers. GST isoenzymes from roach had higher activity to biotransform MC-LR (conversion rate ranging up to 268 ng MC-LR min^? 1 mL^? 1 hepatic enzyme) than that isolated from silver carp. Without any prior contact to MC-LR or another GST inducer, roach seems to be better equipped for microcystin biotransformation than silver carp.     

Selective elution of rodent glutathione S-transferases and glyoxalase I from the S-hexyglutathione-Sepharose affinity matrix.

1. The major hepatic glutathione S-transferases (GSTs) from gerbil, guinea-pig, hamster, mouse and rat comprise Ya- (Mr 25,500-25,800), Yb- (Mr 26,100-26,400), Yc- (Mr 27,000-27,500) and Yf- (Mr 24,800) type subunits. 2. In all rodent species the GST subunits possess characteristic affinities for S-hexyglutathione-Sepharose and are eluted at distinct positions when a gradient of counter-ligand is employed to develop this affinity gel. The enzymes that bind to this matrix can be eluted, according to their subunit composition, in the order Ya-, Yc-, Yf- and Yb-containing GST; glyoxalase I, also retained by S-hexylglutathione-Sepharose, is eluted after the major GST YbYb peak. 3. Conditions are also described for the isocratic affinity elution of S-hexylglutathione-Sepharose that allow rat GST to be divided into four separate fractions (pools 1-4). A further fraction (pool 5) can be prepared from material that does not bind S-hexylglutathione-Sepharose and is obtained by chromatography on glutathione-Sepharose. 4. The sequential use of S-hexylglutathione-Sepharose and glutathione-Sepharose has facilitated the isolation of novel GSTs by enriching the various affinity-purified fractions with different subunits. This strategy allowed the Yk (Mr 25,000) and Yo (Mr 26,500) subunits from rat testis as well as Y1 (Mr 25,700) from rat kidney to be rapidly purified. 5. The binding properties of GST subunits for S-hexylglutathione-Sepharose have been compared with their Km values for GSH. The elution order from this matrix is inversely related to the Km value. The GSTs that do not bind to S-hexylglutathione-Sepharose have considerably higher Km values for GSH (i.e. greater than 2.0 mM) than do those enzymes that readily bind to the affinity gel (i.e. 0.13-0.77 mM). GST YkYk and YoYo, which have weak affinities for S-hexylglutathione-Sepharose, possess intermediate Km values for GSH of 1.0 and 1.2 mM respectively.     

Purification and characterization of glutathione S-transferases of human kidney.

Several forms of glutathione S-transferase (GST) are present in human kidney, and the overall isoenzyme pattern of kidney differs significantly from those of other human tissues. All the three major classes of GST isoenzymes (alpha, mu and pi) are present in significant amounts in kidney, indicating that GST1, GST2 and GST3 gene loci are expressed in this tissue. More than one form of GST is present in each of these classes of enzymes, and individual variations are observed for these classes. The structural, immunological and functional properties of GST isoenzymes of three classes differ significantly from each other, whereas the isoenzymes belonging to the same class have similar properties. All the cationic GST isoenzymes of human kidney except for GST 9.1 are heterodimers of 26,500-Mr and 24,500-Mr subunits. GST 9.1 is a dimer of 24,500-Mr subunits. All the cationic isoenzymes of kidney GST cross-react with antibodies raised against a mixture of GST alpha, beta, gamma, delta and epsilon isoenzymes of liver. GST 6.6 and GST 5.5 of kidney are dimers of 26,500-Mr subunits and are immunologically similar to GST psi of liver. Unlike other human tissues, kidney has at least two isoenzymes (pI 4.7 and 4.9) associated with the GST3 locus. Both these isoenzymes are dimers of 22,500-Mr subunits and are immunologically similar to GST pi of placenta. Some of the isoenzymes of kidney do not correspond to known GST isoenzymes from other human tissues and may be specific to this tissue.     

Selective induction of glutathione S-transferase subunits in wheat plants exposed to the herbicide acifluorfen

Exposure to the herbicide acifluorfen resulted in marked increase of glutathione S-transferase (GST) enzyme activity in wheat seedlings, primarily in shoot tissues. From the six major, constitutively expressed GST subunits found in untreated wheat shoots subunits 2 and 3 were selectively induced by acifluorfen. No new subunit could be detected. The induced subunits belong to those GST isoenzymes, which metabolize diphenyl ether herbicides.     

Anionic and neutral glutathione S-transferase isoenzymes in the freshwater worm Tubifex tubifex (O.F.M.)

The anionic and the neutral glutathione S-transferase (GST) isoenzymes from the freshwater worm Tubifex tubifex (O.F.M.) were separated in one step by chromatofocusing on a Polybuffer exchanger 94 column, eluted with Polybuffer 74. Their pI values ranged between 4.12 and 6.98, and their molecular weight between 30 000 and 38 400. The apparent Km values towards glutathione and 1-chloro-2,4-dinitrobenzene were also determined. The high number of non-cationic GST isoenzymes is unusual. Tubifex worms seems well equipped for attacking environmental pollutants.     

Anti-SLA seropositive autoimmune hepatitis sera recognize distinct subunits of glutathione S-transferase: high prevalence of the Ya autoantigen.

We have recently reported that anti-SLA seropositive autoimmune hepatitis (AIH) patients develop autoantibodies against glutathione S-transferase (GST). GSTs are multifunctional enzymes mediating hepatic detoxification of cytotoxic and genotoxic compounds and are also involved in biliary secretion. We have observed varying reactivity of individual AIH sera towards several GST isoenzymes. Since the GST subunits have very similar molar masses and therefore are not satisfactorily resolved by one-dimensional gel electrophoresis, we have performed their fractionation by reverse-phase high performance liquid chromatography (HPLC) to better separate the individual GST isoenzymes. 4 individual GST subunits were isolated as judged by electrophoretic analysis of the 4 distinct peaks. The identity of isolated proteins was unequivocally determined by protein sequencing. Isolated subtypes were loaded on 15% SDS gels and blotted. Immunoblotting was performed with eleven anti-SLA positive sera that displayed differential reactivity with total GSTs. Fractionation of the GSTs by HPLC did not impair their ability to react with specific autoantibodies. Interestingly, the majority of GST-positive AIH sera reacted with one or two GST subtypes, only two sera recognized 3 subunits. Ya was most prevalent autoantigen. Autoantibodies against Yb2 were detected solely in one serum. This pattern of reactivity indicates that individual patients' sera discriminate between GST subunits despite their sequence homology. It is well known that the GST variants differ within their amino-terminal part while the residual moiety is highly conserved. It would suggest that autoantibodies recognize distinct epitopes located within amino-terminus of individual GST variants.     

Comparative studies on the isoenzymes of glutathione S-transferase of rat brain and other tissues

Six forms of glutathione S-transferase (GST) designated as GST 9.3, GST 7.5, GST 6.6, GST 6.1, GST 5.7 and GST 4.9 have been purified to homogeneity from rat brain. All GST isoenzymes of rat brain are apparent homodimers of one of the three type subunits, Ya, Yb, or Yc. More than 60% of total GST activity of rat brain GST activity is associated with the isoenzymes containing only the Yb type of subunits. In these respects brain GST isoenzymes differ from those of lung and liver. The Ya, Yb, and Yc type subunits of brain GST are immunologically similar to the corresponding subunits of liver and lung GST. The isoelectric points and kinetic properties of the Yb type subunit dimers in brain are strikingly different from those of the Yb type dimers present among liver GST isoenzymes indicating subtle differences between these subunits of brain and liver.     

Expression of human glutathione S-transferase 2 in Escherichia coli. Immunological comparison with the basic glutathione S-transferases isoenzymes from human liver.

A plasmid, termed pTacGST2, which contains the complete coding sequence of a GST2 (glutathione S-transferase 2) subunit and permits the expression of the protein in Escherichia coli was constructed. The expressed protein had the same subunit Mr as the enzyme from normal human liver and retained its catalytic function with both GST and glutathione peroxidase activity. Antiserum raised against the bacterially synthesized protein cross-reacted with all the basic GST isoenzymes in human liver. The electrophoretic mobility in agarose of the bacterially expressed isoenzyme suggested that its pI is identical with that of the cationic isoenzyme from human liver previously termed GST2 type 1. The available evidence suggests that the three common cationic isoenzymes found in human liver are the products of two very similar gene loci.     

Two glutathione S-transferase isoenzymes purified from Bulinus truncatus (Gastropoda: Planorbidae)

We purified and characterized two major glutathione S-transferase isoenzymes (GST2 and GST3) from snail Bulinus truncatus (Mollusca, Gastropoda, Planorbidae) tissue. The Km with respect to 1-chloro-2, 4-dinitrobenzene (CDNB) for both isoenzymes was increased as the pH decreased. Km of both isoenzymes with respect to glutathione (GSH) doubled when the pH was increased from 6.0 to 6.5. Acid inactivated GST2 and GST3 and the two enzymes were almost inactive at pH 3.5. However, they retain the full activity for at least 20 h when incubated at pH between 6.0 and 9.0. The optimum temperature was 45 degrees C for GST2 and 50 degrees C for GST3. The half lifetime at 50 degrees C was 70 min and 45 min for GST2 and GST3 isoenzymes, respectively. Addition of 5 mM GSH to the incubation buffer increased the half life of both isoenzymes more than fourfold. The activation energy for catalyzing the conjugation of CDNB was 1.826 and 3.435 kcal/mol for GST2 and GST3, respectively. I50 values for Cibacron blue, bromosulphophthalein, indocyanine green, hematin and ethacrynic acid were 0.76 microM, 47.9 microM, 7.59 microM, 0.03 microM and 0.79 microM for GST2, and 0.479 microM, 79.4 microM, 89.1 microM, 32.4 microM and 1.15 microM for GST3, respectively. Cibacron blue and indocyanine green were non-competitive inhibitors, while hematin was a mixed inhibitor. Bromosulphophthalein was found to be a competitive inhibitor for GST2 and a mixed inhibitor for GST3.     

Expression of glutathione transferase isoenzymes in the porcine ovary in relationship to follicular maturation and luteinization

The expression of different isoenzymes of glutathione transferase (GST), i.e. the cytosolic subunits GSTA1/A2, A3, A4, A5, M1/2, M2 and P1, T2, and the microsomal GST in follicles of different sizes and in corpora lutea from porcine ovary, was investigated by Western blotting. No immunoreactivity was obtained with anti-rat GSTT2 or anti-rat microsomal GST polyclonal antibodies. In contrast, GSTA1/A2, A3, A4, A5, M1/2, M2 and P1 are all expressed in the cytosol from porcine ovaries. In general, the highest levels of these GST isoenzymes were present in the cytosol from corpora lutea, in agreement with measurements of activity towards 1-chloro-2,4-dinitrobenzene. Immunoreactivity with anti-rat GSTP1 was only obtained with follicles. The cytosolic GSTs from follicles and corpora lutea were affinity purified on glutathione-Sepharose and separated by reversed-phase high-performance liquid chromatography in order to quantitate the different subunits. A peak corresponding to the class pi subunit was present in follicles. This peak was also seen with corpora lutea, although at very low level. There were four peaks containing class mu subunits. The remaining peaks were concluded to contain the class alpha subunits, except for two peaks which are suggested to contain proteins other than GSTs. The levels of the different subunits were quantitated on the basis of the areas under the peaks and the relative amounts in follicles of different sizes and in corpora lutea corresponded well with the Western blot analysis.     

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.     

Differential activities of rat and human lung glutathione S-transferase isoenzymes towards benzo(a)pyrene epoxides

The isoenzymes of human and rat lung glutathione S-transferase (GST) differ among themselves in their activities towards the epoxides of benzo(a)pyrene (BP). The Ya' and Yc-type subunits of rat lung GST exhibit maximum activities towards BP-4,5-oxide and BP-7,8-oxide suggesting that these two subunits are preferentially involved in the detoxification of highly reactive epoxides and diol-epoxides of polycyclic aromatic hydrocarbons (PAH). The studies with human lung GST isoenzymes indicate that BP-4,5-oxide, and BP-7,8-oxide are preferred substrates for the cationic (pI 8.3) form of the enzyme. Identification of compounds which can selectively induce these isoenzymes of GST could prove useful as inhibitors of PAH induced neoplasia.     

Do the major human glutathione S-transferases have fatty acid ethyl ester synthase activity?

Two fatty acid ethyl ester (FAEE) synthase isoenzymes purified from human myocardium were reported to be glutathione S-transferases (GST) [(1989) Proc. Natl. Acad. Sci. USA 86, 4470-4473; and (1989) J. Clin. Invest. 84, 1942-1946]. In the present study, the FAEE synthase activity of several purified and well characterized human GSTs were examined with ethanol and [14C]oleic acid as substrates. Three isoenzymes, GST1, GST2 and GST3 which are members of the evolutionary classes mu, alpha, and pi, respectively, were studied and failed to show any significant synthesis of FAEE after 45 min incubation at 37 degrees C. FAEE synthase activity and GST3 activity in human placental extracts can be readily separated by ion exchange chromatography on DEAE cellulose. Thus the results show that FAEE synthase activity is not a feature of the major GSTs found in human tissues. The two FAEE synthase isoenzymes isolated by Bora et al. may have been co-purified with GST isoenzymes or these FAEE synthases may be members of the GST super family that have low specific activity in conventional GST assays and have not been previously described.     

Age-development and inducibility of hepatic glutathione S-transferase activities in mice, rats, rabbits and guinea-pigs

Hepatic glutathione S-transferase (GST) activities towards 1-chloro-3,4-dinitrobenzene (DNCB), 3,4-dichloronitrobenzene (DCNB), sulfobromophthalein (BSP), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), trans-4-phenyl-3-buten-2-one (TPBO) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) were determined in mice, rats, rabbits and guinea-pigs during ageing and after pretreatment with enzyme inducers. Variations were observed in the developmental patterns and in the phenobarbital-, benzo(a)pyrene-, pregnenolone-16 alpha-carbonitrile-, butylated hydroxyanisole-, trans-stilbene oxide-inducibility of hepatic GST activities in the same species towards different substrates. For example, in rats GST activities for EA, DCNB and TPBO increased respectively, 2.3-, 4.8- and 25-fold during age-development, and after treatment with TSO 1.2-, 3.6- and 1.3-fold. Species differences were found in the maturation and in the inducibility of GST activities. For instance, GST activity toward EA at birth is mature in guinea pigs but not in the other species; phenobarbital treatment increased GST activities in mice and rats but not in rabbits and guinea-pigs; treatment with trans-stilbene oxide enhanced GST activity for TPBO 4.5-fold in mice but not at all in rats. It is concluded that hepatic glutathione conjugation exhibits functional heterogeneity which may be due to species dependent variations in the responsiveness of GST isoenzymes to endogenous and exogenous influences.     

Differential expression of alpha, mu and pi classes of isozymes of glutathione S-transferase in bovine lens, cornea, and retina

Isozyme characterization of glutathione S-transferase (GST) isolated from bovine ocular tissue was undertaken. Two isozymes of lens, GST 7.4 and GST 5.6, were isolated and found to be homodimers of a Mr 23,500 subunit. Amino acid sequence analysis of a 20-residue region of the amino terminus was identical for both isozymes and was identical to GST psi and GST mu of human liver. Antibodies raised against GST psi cross-reacted with both lens isozymes. Although lens GST 5.6 and GST 7.4 demonstrated chemical and immunological relatedness, they were distinctly different as evidenced by their pI and comparative peptide fingerprint. A corneal isozyme, GST 7.2, was also isolated and established to be a homodimer of Mr 24,500 subunits. Sequence analysis of the amino-terminal region indicated it to be about 67% identical with the GST pi isozyme of human placenta. Antibodies raised against GST pi cross-reacted with cornea GST 7.2. Another corneal isozyme, GST 8.7, was found to be homodimer of Mr 27,000 subunits. Sequence analysis revealed it to have a blocked amino-terminus. GST 8.7 immunologically cross-reacted with the antibodies raised against cationic isozymes of human liver indicating it to be of the alpha class. Two isozymes of retina, GST 6.8 and GST 6.3, were isolated and identified to be heterodimers of subunits of Mr 23,500 and 24,500. Amino-terminal sequence analysis gave identical results for both retina GST 6.8 and GST 6.3. The sequence analysis of the Mr 23,500 subunit was identical to that obtained for lens GSTs. Similarly, sequence analysis of the Mr 24,500 subunit was identical to that obtained for the cornea GST 7.2 isozyme. Both the retina isozymes cross-reacted with antibodies raised against human GST psi as well as GST pi. The results of these studies indicated that all three major classes of GST isozymes were expressed in bovine eye but the GST genes were differentially expressed in lens, cornea, and retina. In lens only the mu class of GST was expressed, whereas cornea expressed alpha and pi classes and retina expressed mu and pi classes of GST isozymes.