Isolation and characterization of six human hepatic isometallothioneins.

Human brain contains one cationic (pI8.3) and two anionic (pI5.5 and 4.6) forms of glutathione S-transferase. The cationic form (pI8.3) and the less-anionic form (pI5.5) do not correspond to any of the glutathione S-transferases previously characterized in human tissues. Both of these forms are dimers of 26500-Mr subunits; however, immunological and catalytic properties indicate that these two enzyme forms are different from each other. The cationic form (pI8.3) cross-reacts with antibodies raised against cationic glutathione S-transferases of human liver, whereas the anionic form (pI5.5) does not. Additionally, only the cationic form expresses glutathione peroxidase activity. The other anionic form (pI4.6) is a dimer of 24500-Mr and 22500-Mr subunits. Two-dimensional gel electrophoresis demonstrates that there are three types of 26500-Mr subunits, two types of 24500-Mr subunits and two types of 22500-Mr subunits present in the glutathione S-transferases of human brain.     

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.     

Rat lung glutathione S-transferases: subunit structure and the interrelationship with the liver enzymes

Six forms of glutathione S-transferases designated as GSH S-transferase I (pI 8.8), II (pI 7.2), III (pI 6.8), IV (pI 6.0), V (pI 5.3) and VI (pI 4.8) have been purified from rat lung. GSH S-transferase I (pI 8.8) is a homodimer of Mr 25,000 subunits; GSH S-transferases II (pI 7.2) and VI (pI 4.8) are homodimers of Mr 22,000 subunits; and GSH S-transferases III (pI 6.8), IV (pI 6.0) and V (pI 5.3) are dimers composed of Mr 23,500 and 22,000 subunits. Immunological properties, peptide fragmentation analysis, and substrate specificity data indicate that Mr 22,000, 23,500 and 25,000, are distinct from each other and correspond to Ya, Yb, and Yc subunits, respectively, of rat liver.     

Use of immuno-blot techniques to discriminate between the glutathione S-transferase Yf, Yk, Ya, Yn/Yb and Yc subunits and to study their distribution in extrahepatic tissues. Evidence for three immunochemically distinct groups of transferase in the rat.

The glutathione S-transferases are dimeric enzymes whose subunits can be defined by their mobility during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis as Yf (Mr 24,500), Yk (Mr 25,000), Ya (Mr 25,500), Yn (Mr 26,500), Yb1 (Mr 27,000), Yb2 (Mr 27,000) and Yc (Mr 28,500) [Hayes (1986) Biochem. J. 233, 789-798]. Antisera were raised against each of these subunits and their specificities assessed by immuno-blotting. The transferases in extrahepatic tissues were purified by using, sequentially, S-hexylglutathione and glutathione affinity chromatography. Immune-blotting was employed to identify individual transferase polypeptides in the enzyme pools from various organs. The immuno-blots showed marked tissue-specific expression of transferase subunits. In contrast with other subunits, the Yk subunit showed poor affinity for S-hexylglutathione-Sepharose 6B in all tissues examined, and subsequent use of glutathione and glutathione affinity chromatography. Immuno-blotting was employed to identify a new cytosolic polypeptide, or polypeptides, immunochemically related to the Yk subunit but with an electrophoretic mobility similar to that of the Yc subunit; high concentrations of the new polypeptide(s) are present in colon, an organ that lacks Yc.     

Identification of a basic hybrid glutathione S-transferase from human liver. Glutathione S-transferase delta is composed of two distinct subunits (B1 and B2).

The purification of a hybrid glutathione S-transferase (B1 B2) from human liver is described. This enzyme has an isoelectric point of 8.75 and the B1 and B2 subunits are distinguishable immunologically and are ionically distinct. Hybridization experiments demonstrated that B1 B1 and B2 B2 could be resolved by CM-cellulose chromatography and have pI values of 8.9 and 8.4 respectively. Transferase B1 B2, and the two homodimers from which it is formed, are electrophoretically and immunochemically distinct from the neutral enzyme (transferase mu) and two acidic enzymes (transferases rho and lambda). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that B1 and B2 both have an Mr of 26 000, whereas, in contrast, transferase mu comprises subunits of Mr 27 000 and transferases rho and lambda both comprise subunits of Mr 24 500. Antisera raised against B1 or B2 monomers did not cross-react with the neutral or acidic glutathione S-transferases. The identity of transferase B1 B2 with glutathione S-transferase delta prepared by the method of Kamisaka, Habig, Ketley, Arias & Jakoby [(1975) Eur. J. Biochem. 60, 153-161] has been demonstrated, as well as its relationship to other previously described transferases.     

Rat lung glutathione S-transferases. Evidence for two distinct types of 22000-Mr subunits.

Two immunologically distinct types of 22000-Mr subunits are present in rat lung glutathione S-transferases. One of these subunits is probably similar to Ya subunits of rat liver glutathione S-transferases, whereas the other subunit Ya' is immunologically distinct. Glutathione S-transferase II (pI7.2) of rat lung is a heterodimer (YaYa') of these subunits, and glutathione S-transferase VI (pI4.8) of rat lung is a homodimer of Ya' subunits. On hybridization in vitro of the subunits of glutathione S-transferase II of rat lung three active dimers having pI values 9.4, 7.2 and 4.8 are obtained. Immunological properties and substrate specificities indicate that the hybridized enzymes having pI7.2 and 4.8 correspond to glutathione S-transferases II and VI of rat lung respectively.     

Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree).

A hitherto unknown cytosolic glutathione S-transferase from rat liver was discovered and a method developed for its purification to apparent homogeneity. This enzyme had several properties that distinguished it from other glutathione S-transferases, and it was named glutathione S-transferase X. The purification procedure involved DEAE-cellulose chromatography, (NH4)2SO4 precipitation, affinity chromatography on Sepharose 4B to which glutathione was coupled and CM-cellulose chromatography, and allowed the isolation of glutathione S-transferases X, A, B and C in relatively large quantities suitable for the investigation of the toxicological role of these enzymes. Like glutathione S-transferase M, but unlike glutathione S-transferases AA, A, B, C, D and E, glutathione S-transferase X was retained on DEAE-cellulose. The end product, which was purified from rat liver 20 000 g supernatant about 50-fold, as determined with 1-chloro-2,4-dinitrobenzene as substrate and about 90-fold with the 1,2-dichloro-4-nitrobenzene as substrate, was judged to be homogeneous by several criteria, including sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, isoelectric focusing and immunoelectrophoresis. Results from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration indicated that transferase X was a dimer with Mr about 45 000 composed of subunits with Mr 23 500. The isoelectric point of glutathione S-transferase X was 6.9, which is different from those of most of the other glutathione S-transferases (AA, A, B and C). The amino acid composition of transferase X was similar to that of transferase C. Immunoelectrophoresis of glutathione S-transferases A, C and X and precipitation of various combinations of these antigens by antisera raised against glutathione S-transferase X or C revealed that the glutathione S-transferases A, C and X have different electrophoretic mobilities, and indicated that transferase X is immunologically similar to transferase C, less similar to transferase A and not cross-reactive to transferases B and E. In contrast with transferases B and AA, glutathione S-transferase X did not bind cholic acid, which, together with the determination of the Mr, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyse the reaction of menaphthyl sulphate with glutathione, and was in this respect dissimilar to glutathione S-transferase M; however, it conjugated 1,2-dichloro-4-nitrobenzene very rapidly, in contrast with transferases AA, B, D and E, which were nearly inactive towards that substrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Two immunologically distinct Yc-type subunits are present in rat lung glutathione S-transferases.

Two types of 25 000-Mr subunits are present in rat lung glutathione S-transferase I (pI 8.8). These subunits, designated Yc and Yc', are immunologically and functionally distinct from each other. The homodimers YcYc (pI 10.4) and Yc'Yc' (pI 7.6) obtained by hybridization in vitro of the two subunits of glutathione S-transferase I (pI 8.8) were isolated and characterized. Results of these studies indicate that only the Yc subunits express glutathione peroxidase activity and cross-react with the antibodies raised against glutathione S-transferase B (YaYc) or rat liver. The Yc' subunits do not express glutathione peroxidase activity and do not cross-react with the antibodies raised against glutathione S-transferase B of rat liver. The amino acid compositions of these two subunits are also different. These two subunits can also be separated by the two-dimensional gel electrophoresis of glutathione S-transferase I (pI 8.8) of rat lung.     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

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.     

Sex-specific constitutive expression of the pre-neoplastic marker glutathione S-transferase, YfYf, in mouse liver.

Hepatic glutathione S-transferase isoenzyme content has been investigated in both sexes of three inbred strains of mice (DBA/2, C3H/He, C57BL6). A polypeptide (Mr 24,800), which is immunologically related to Yf purified from rat lung, was found to be expressed as a major form in all male mouse livers but represented only a minor enzyme form in female mouse liver. Glutathione S-transferases comprising subunits with molecular masses of 25,800 (Ya) or 26,400 (Yb) were present in males and females of the three strains under investigation. Cytosolic isoenzymes from all strains and sexes were purified to apparent homogeneity and no significant inter-strain differences in the properties of the individual forms were observed. In addition, no differences were detected in the microsomal glutathione S-transferase content of the different strains or sexes.     

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.     

Tissue-specific expression of the rat glutathione S-transferases

Tissue-specific patterns of rat glutathione S-transferase expression have been demonstrated by in vitro translation of purified poly(A) RNAs and by protein purification. Poly(A) RNAs from six rat tissues including heart, kidney, liver, lung, spleen, and testis were used to program in vitro translation with the rabbit reticulocyte lysate system and [35S]methionine. The glutathione S-transferase subunits synthesized in vitro were purified from the translation products by affinity chromatography on S-hexylglutathione-linked Sepharose 6B columns. The affinity bound fractions were analyzed by Na dodecyl SO4-polyacrylamide gel electrophoresis and fluorography. A subunit of Mr = 22,000 detected in the in vitro translation products of poly(A) RNAs from heart, kidney, lung, spleen, and testis is missing from the translation products of liver poly(A) RNAs. This Mr = 22,000 subunit is present only in the anionic glutathione S-transferase fraction purified from rat heart, kidney, lung, spleen, and testis. Purified anionic glutathione S-transferase from rat liver does not contain this subunit. The relative specific activities toward a dozen different substrates also demonstrate the nonidentity between liver and kidney anionic glutathione S-transferases. In addition, among the glutathione S-transferase subunits expressed in the liver, some of them could not be detected in the other tissues investigated. Our results indicate that tissue-specific expression of rat glutathione S-transferases may occur pretranslationally.     

Glutathione S-transferase in chemotherapy resistance and in carcinogenesis.

Cytosolic glutathione S-transferases are composed of two monomeric subunits. These monomers are the products of different gene families designated alpha, mu, and pi. Dimerization yields either homodimeric or heterodimeric holoenzymes within the same family. The members of this complex group of proteins have been linked to the detoxification of environmental chemicals and carcinogens, and have been shown to be overexpressed in normal and tumor cells following exposure to cytotoxic drugs. They also are overexpressed in carcinogen-induced rat liver preneoplastic nodules in rat liver. In all of these cases, the changes in expression of glutathione S-transferases are paralleled by increased resistance to cytotoxic chemicals. The degree of resistance is related to the substrate specificity of the isozyme. The relationship of the glutathione S-transferase genes to drug resistance has been directly demonstrated by gene transfer studies, where cDNAs encoding the various subunits of glutathione S-transferase have been transfected into a variety of cell types. This review discusses the results of numerous studies that associate resistance to alkylating agents with overexpression of protective detoxifying glutathione S-transferase enzymes.     

Anomalous electrophoretic behaviour of the glutathione S-transferase Ya and Yk subunits isolated from man and rodents. A potential pitfall for nomenclature.

GSH S-transferases are dimeric enzymes. The subunits in the rat are resolved into six types, designated Yf, Yk, Ya, Yn, Yb and Yc, by discontinuous SDS/polyacrylamide-gel electrophoresis [Hayes (1986) Biochem. J. 233, 789-798]. The relative electrophoretic mobility of the Ya and Yk subunits is dependent on the amount of cross-linker (NN'-methylenebisacrylamide) in the resolving gel. At low degrees of cross-linking, CBis 0.6% (w/w), the Yk and Ya subunits possess a faster anodal mobility than do the Yf, Yn, Yb and Yc subunits (i.e. order of mobility Yk greater than Ya greater than Yf greater than Yn greater than Yb greater than Yc), whereas at higher degrees of cross-linking, CBis 5.0% (w/w), Yf subunits possess the fastest mobility (i.e. order of mobility Yf greater than Yk greater than or equal to Yn greater than Yb greater than or equal to Ya greater than Yc). Resolving gels that contain low concentrations of cross-linker [CBis 0.6% (w/w)] allow the resolution of a hitherto unrecognized polypeptide that is isolated by S-hexyl-GSH-Sepharose affinity chromatography. This new polypeptide, which we have designated Yb, is normally obscured by the main Yb band in resolving gels that comprise concentrations of cross-linker of at least CBis 1.6% (w/w). The Ya- and Yb-type subunits in guinea pig, mouse, hamster and man were identified by immuno-blotting and their apparent Mr values in different electrophoresis systems were determined. The Ya subunits in all species studied possess a variable cross-linker-dependent mobility during electrophoresis. Since the transferase subunits are currently classified according to their mobilities during SDS/polyacrylamide-gel electrophoresis, it is apparent that the variable electrophoretic behaviour of the Ya and Yk subunits may lead to the mis-identification of enzymes.     

Purification and characterization of glutathione S-transferase isozymes in dog lens.

1. Two isozymes of glutathione S-transferase (GST-dl1 and GST-dl2) were purified to homogeneity from dog lens. 2. The subunit size and the isoelectric point were determined to be 24,000 and > pI 9.5 for GST-dl1 and 22,000 and pI 8.1 for GST-dl2. 3. It was judged that GST-dl1 is a class alpha enzyme and GST-dl2 belongs to class pi on the basis of their immunological properties and N-terminal amino acid sequences. 4. The expression pattern of glutathione S-transferase isoenzymes in dog lens is different from that in pig, rat and bovine lenses.     

Interrelationship between cationic and anionic forms of glutathione S-transferases of bovine ocular lens.

Since the eye is constantly exposed to potentially damaging chemical compounds present in the atmosphere and vascular system, we investigated the physiological role of glutathione S-transferase (GSH S-transferase) in detoxification mechanisms operative in the ocular lens. We have purified an anionic and a cationic GSH S-transferase from the bovine lens to homogeneity through a combination of gel filtration, ion-exchange and affinity chromatography. The anionic (pI 5.6) and cationic (pI 7.4) S-transferases were found to have distinct kinetic parameters (apparent Km and Vmax. pH optimum and energy of activation). However, both species were demonstrated to have similar molecular weights and amino acid compositions. Double-immunodiffusion and immunotitration studies showed that both lens S-transferases were immunologically similar. The very close similarity in amino acid compositions and immunological properties strongly indicates that these two transferases either originate from the same gene or at least share common antigenic determinants and originate from similar genes. The bovine lens GSH S-transferases had no glutathione peroxidase activity with either t-butyl hydroperoxide or cumene hydroperoxide as substrate. However, the antibody raised against the homogeneous anionic glutathione S-transferase from the bovine lens was found to precipitate both glutathione S-transferase and glutathione peroxidase activities out of solution in the supernatant of a crude bovine liver homogenate.     

Identification of a novel glutathione transferase in human skin homologous with class alpha glutathione transferase 2-2 in the rat.

Six forms of glutathione transferase with pI values of 4.6, 5.9, 6.8, 7.1, 8.5 and 9.9 have been isolated from the cytosol fraction of normal skin from three human subjects. The three most abundant enzymes were an acidic Class Pi transferase (pI 4.6; apparent subunit Mr 23,000), a basic Class Alpha transferase (pI 8.5; apparent subunit Mr 24,000) and an even more basic glutathione transferase of Class Alpha (pI 9.9; apparent subunit Mr 26,500). The last enzyme, which was previously unknown, accounts for 10-20% of the glutathione transferase in human skin. The novel transferase showed greater similarities with rat glutathione transferase 2-2, another Class Alpha enzyme, than with any other known transferase irrespective of species. The most striking similarities included reactions with antibodies, amino acid compositions and identical N-terminal amino acid sequences (16 residues). The close relationship between the human most basic and the rat glutathione transferase 2-2 supports the classification of the transferases previously proposed and indicates that the similarities between enzymes isolated from different species are more extensive than had been assumed previously.     

Different forms of human liver glutathione S-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits.

Four immunologically distinct subunits were characterized in glutathione (GSH) S-transferases of human liver. Five cationic enzymes (pI 8.9, 8.5, 8.3, 8.2 and 8.0) have an apparently similar subunit composition, and are dimers of 26 500-Mr (A) and 24 500-Mr (B) subunits. A neutral enzyme, pI 6.8, is a dimer of B-type subunits. One of the anionic enzymes, pI 5.5, is also a dimer of 26 500-Mr subunits. However, the 26 500-Mr subunits of this anionic enzyme form are immunologically distinct from the A subunits of the cationic enzymes, and have been designated as A'. Immunoabsorption studies with the neutral enzyme, BB, and the antibodies raised against the cationic enzymes (AB) indicate that A and B subunits are immunologically distinct. Hybridization in vitro of the A and B subunits of the cationic enzymes (AB) results in the expected binary combinations of AA, AB and BB. Studies with the hybridized enzyme forms indicate that only the A subunits express GSH peroxidase activity. A' subunits have maximum affinity for p-nitrobenzyl chloride and p-nitrophenyl acetate, and the B subunits have highest activity towards 1-chloro-2,4-dinitrobenzene. The other anionic form, pI 4.5, present in liver is a heterodimer of 22 500-Mr (C) and B subunits. The C subunits of this enzyme are probably the same as the 22 500-Mr subunits present in human lung and placental GSH transferases. The distinct immunological nature of B and C subunits was also demonstrated by immunoaffinity and subunit-hybridization studies. The results of two-dimensional polyacrylamide-gel-electrophoretic analyses indicate that in human liver GSH transferases, three charge isomers of Mr 26 500 (A type), two charge isomers of Mr 24 500 (B type) and two charge isomers of Mr 22 500 (C type) subunits are present.