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.     

Expression of glyoxalase, glutathione peroxidase and glutathione S-transferase isoenzymes in different bovine tissues

(1) The tissue-specific expression of various glutathione-dependent enzymes, including glutathione S-transferase (GST), glutathione peroxidase and glyoxalase I, has been studied in bovine adrenals, brain, heart, kidney, liver, lung and spleen. Of the organs studied, liver was found to possess the greatest GST and glyoxalase I activity, and spleen the greatest glutathione peroxidase activity. The adrenals contained large amounts of these glutathione-dependent enzymes, but significant differences were observed between the cortex and medulla. (2) GST and glyoxalase I activity were isolated by S-hexylglutathione affinity chromatography. Glyoxalase I was found in all the organs examined, but GST exhibited marked tissue-specific expression. (3) The alpha, mu and pi classes of GST (i.e., those that comprise respectively Ya/Yc, Yb/Yn and Yf subunits) were all identified in bovine tissues. However, the Ya and Yc subunits of the alpha class GST were not co-ordinately regulated nor were the Yb and Yn subunits of the mu class GST. (4) Bovine Ya subunits (25.5-25.7 kDa) were detected in the adrenal, liver and kidney, but not in brain, heart, lung or spleen. The Yc subunit (26.4 kDa) was expressed in all those organs which expressed the Ya subunit, but was also found in lung. The mu class Yb (27.0 kDa) and Yn (26.1 kDa) subunits were present in all organs; however, brain, lung and spleen contained significantly more Yn than Yb type subunits. The pi class Yf subunit (24.8 kDa) was detected in large amounts in the adrenals, brain, heart, lung and spleen, but not in kidney or liver. (5) Gradient affinity elution of S-hexylglutathione-Sepharose showed that the bovine proteins that bind to this matrix elute in the order Ya/Yc, Yf, Yb/Yn and glyoxalase I. (6) In conclusion, the present investigation has shown that bovine GST are much more complex than previously supposed; Asaoka (J. Biochem. 95 (1984) 685-696) reported the purification of mu class GST but neither alpha nor pi class GST were isolated.     

The development of glutathione S-transferase subunits in rat liver. Sensitive detection of the major subunit forms of rat glutathione S-transferase by using an e.l.i.s.a. method.

The development of the subunits of glutathione S-transferase in rat liver shows that there is a co-ordinated development of the Ya, Yb1, Yb2 and Yc subunits but that the Yf and Yk subunits show unique patterns of development. The Yk subunit is the only form that is expressed at relatively high levels during the foetal period as well as during the adult period. In contrast with all other forms, the Yf subunit in the rat declines rapidly during the last few days before parturition and is virtually undetectable in hepatocytes of adult animals. The expression of the Yf subunit in foetal liver presents a 'patchy' appearance that is similar to that induced by the administration of lead acetate and may reflect cell-cycle-associated regulation of expression.     

Inhibition of hepatic and extrahepatic glutathione S-transferases by primary and secondary bile acids.

Glutathione S-transferases are a complex family of dimeric proteins that play a dual role in cellular detoxification; they catalyse the first step in the synthesis of mercapturic acids, and they bind potentially harmful non-substrate ligands. Bile acids are quantitatively the major group of ligands encountered by the glutathione S-transferases. The enzymes from rat liver comprise Yk (Mr 25 000), Ya (Mr 25 500), Yn (Mr 26 500), Yb1, Yb2 (both Mr 27 000) and Yc (Mr 28 500) monomers. Although bile acids inhibited the catalytic activity of all transferases studied, the concentration of a particular bile acid required to produce 50% inhibition (I50) varies considerably. A comparison of the I50 values obtained with lithocholate (monohydroxylated), chenodeoxycholate (dihydroxylated) and cholate (trihydroxylated) showed that, in contrast with all other transferase monomers, the Ya subunit possesses a relatively hydrophobic bile-acid-binding site. The I50 values obtained with lithocholate and lithocholate 3-sulphate showed that only the Ya subunit is inhibited more effectively by lithocholate than by its sulphate ester. Other subunits (Yk, Yn, Yb1 and Yb2) were inhibited more by lithocholate 3-sulphate than by lithocholate, indicating the existence of a significant ionic interaction, in the bile-acid-binding domain, between (an) amino acid residue(s) and the steroid ring A. By contrast, increasing the assay pH from 6.0 to 7.5 decreased the inhibitory effect of all bile acids studied, suggesting that there is little significant ionic interaction between transferase subunits and the carboxy group of bile acids. Under alkaline conditions, low concentrations (sub-micellar) of nonsulphated bile acids activated Yb1, Yb2 and Yc subunits but not Yk, Ya and Yn subunits. The diverse effects of the various bile acids studied on transferase activity enables these ligands to be used to help establish the quaternary structure of individual enzymes. Since these inhibitors can discriminate between transferases that appear to be immunochemically identical (e.g. transferases F and L), bile acids can provide information about the subunit composition of forms that cannot otherwise be distinguished.     

Induction and suppression of glutathione transferases by interferon in the mouse

The administration of interferon-alpha/beta to female nude (nu/nu) mice caused significant changes in the levels of the cytosolic hepatic glutathione transferases. Antibodies raised against rat subunits, Ya, Yc, Yb1, Yb2, and Yk, and the subunits of the human transferases, mu (YbYb), lambda (YfYf), and epsilon (B1B1) all reacted with enzymes in the mouse and were used to demonstrate suppression and induction of transferase levels. Western blot analysis followed by semiquantitation by laser scanning showed the Ya, Yb1, Yb2, Yc, Yk, mu, and B1 subunits to be suppressed by 11, 11, 44, 30, 12, 14, and 47%, respectively, by interferon treatment. In contrast to these findings, the Yf subunit was induced 5-7-fold. A concomitant 220% increase was observed in the specific activity of the hepatic cytosol for ethacrynic acid, a substrate for the Yf subunit. Changes in the levels of transferase enzymes in normal and tumor cells may have significant implications when cytotoxic drugs are used in combination with interferons in cancer therapy. The Yf subunit, an enzyme found in human tumors and in placenta (Polidoro, G., Di Mio, C., Del Boccio, G., Zulli, P., and Fererici, G. (1980) Biochem. Pharmacol. 29, 1677-1680) has also been shown to be elevated in hepatic preneoplastic lesions (Kitahara, A., Satoh, K., Nishimura, K., Ishikawa, T., Ruike, K., Sato, K., Tsuda, H., and Ito, N. (1984) Cancer Res. 44, 2698-2703). These data indicate that the Yf subunit represents a potentially important interferon-inducible gene product.     

Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver.

A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].     

Purification and characterization of glutathione S-transferases P, S and N. Isolation from rat liver of Yb1 Yn protein, the existence of which was predicted by subunit hybridization in vitro.

The glutathione S-transferases are dimeric proteins and comprise subunits of Mr 25 500 (Ya), 26 500 (Yn), 27 000 (Yb1 and Yb2) and 28 500 (Yc). Enzymes containing Ya and/or Yc subunits have been isolated as have forms containing binary combinations of Yn, Yb1 and Yb2 subunits. To date only one enzyme, transferase S, has been described that is a YbYn heterodimer [Hayes & Chalmers (1983) Biochem. J. 215, 581-588]; the identity of the Yb monomer found in transferase S has not been reported previously. The identification and isolation of a YnYn dimer (transferase N) from rat testis is now described. This has enabled structural and functional comparisons to be made between Yb1, Yb2 and Yn monomers. Reversible dissociation experiments between the YnYn and Yb1Yb1 homodimers and between the YnYn and Yb2Yb2 homodimers demonstrated that Yn monomers can hybridize with both Yb1 and Yb2 monomers. Reversible dissociation of transferases N and C (Yb1Yb2) showed that both Yb1 and Yb2 monomers can hybridize with Yn monomers under competitive conditions. The hydridization data suggest that transferase S represents the Yb2Yn subunit combination. A knowledge of the elution position from chromatofocusing columns of the Yb1Yn hybrid that was formed in vitro enabled a purification scheme to be devised for an enzyme from rat liver (transferase P) believed to consist of Yb1Yn subunits. A comparison of the chromatographic behaviour of the YnYn, Yb1Yb1 and Yb2Yb2 dimers on chromatofocusing and hydroxyapatite columns with the behaviour of transferases P and S on the same matrices suggests these two enzymes may be identified as the Yb1Yn and Yb2Yn dimers respectively. The catalytic activities and the inhibitory effects of non-substrate ligands on transferases P and S are significantly different and again suggest they comprise Yb1 and Yn subunits and Yb2 and Yn subunits respectively; transferase P exhibits a 6-fold higher specific activity for 1,2-dichloro-4-nitrobenzene than does transferase S, whereas, conversely, transferase S possesses a 9-fold higher specific activity for trans-4-phenylbut-3-en-2-one than does transferase P. The quaternary structure of transferases P and S was verified by using peptide mapping and 'Western blotting' techniques.     

Expression of glutathione S-transferases in rat brains

The tissue-specific expression of glutathione S-transferases (GSTs) in rat brains has been studied by protein purification, in vitro translation of brain poly(A) RNAs, and RNA blot hybridization with cDNA clones of the Ya, Yb, and Yc subunit of rat liver GSTs. Four classes of GST subunits are expressed in rat brains at Mr 28,000 (Yc), Mr 27,000 (Yb), Mr 26,300, and Mr 25,000. The Mr 26,3000 species, or Y beta, has an electrophoretic mobility between that of Ya and Yb, similar to the liver Yn subunit(s) reported by Hayes (Hayes, J. D. (1984) Biochem. J. 224, 839-852). RNA blot hybridization of brain poly(A) RNAs with a liver Yb cDNA probe revealed two RNA species of approximately 1300 and approximately 1100 nucleotides. The band at approximately 1300 nucleotides was absent in liver poly(A) RNAs. The Mr 25,000 species, or Y delta, can be immunoprecipitated by antisera against rat heart and rat testis GSTs, but not by antiserum against rat liver GSTs. Therefore, the Y delta subunit may be related to the "Mr 22,000" subunit reported by Tu et al. (Tu, C.-P.D., Weiss, M.J., Li, N., and Reddy, C. C. (1983) J. Biol. Chem. 258, 4659-4662). The abundant liver GST subunits, Ya, are not expressed in rat brains as demonstrated by electrophoresis of purified brain GSTs and a lack of isomerase activity toward the Ya-specific substrate, delta 5-androstene-3,17-dione. This is apparently because of the absence of Ya mRNA expression prior to RNA processing. The data on the preferential expression of Yc subunits in rat brains, together with the differential phenobarbital inducibility of the Ya subunit(s) in rat liver reported by Pickett et al. (Pickett, C. B., Donohue, A. M., Lu, A. Y. H., and Hales, B. F. (1982) Arch. Biochem. Biophys. 215, 539-543), suggest that the Ya and Yc genes for rat GSTs are two functionally distinct gene families even though they share 68% DNA sequence homology. The expression of multiple GSTs in rat brains suggests that GSTs may be involved in physiological processes other than xenobiotics metabolism.     

Rat liver glutathione S-transferases. Complete nucleotide sequence of a glutathione S-transferase mRNA and the regulation of the Ya, Yb, and Yc mRNAs by 3-methylcholanthrene and phenobarbital

With the use of cDNA probes reverse transcribed from purified glutathione S-transferase mRNA templates, four cDNA clones complementary to transferase mRNAs have been identified and characterized. Two clones, pGTB38 and pGTB34, have cDNA inserts of approximately 950 and 900 base pairs, respectively, and hybridize to a mRNA(s) whose size is approximately 980 nucleotides. In hybrid-select translation experiments, pGTB38 and pGTB34 select mRNAs specific for the Ya and Yc subunits of rat liver glutathione S-transferases. Clone pGTB33, which harbors a truncated cDNA insert, hybrid-selects only the Ya mRNA. All of the clones, pGTB38, pGTB34, and pGTB33, hybrid-select another mRNA which is specific for a polypeptide with an electrophoretic mobility slightly greater than the Ya subunit. The entire nucleotide sequence of the full length clone, pGTB38, has been determined and the complete amino acid sequence of the corresponding polypeptide has been deduced. The mRNA codes for a protein comprising 222 amino acids with Mr = 25,547. We have also identified a cDNA clone complementary to a Yb mRNA of the rat liver glutathione S-transferases. This clone, pGTA/C36, hybrid-selects only Yb mRNA(s) and hybridizes to a mRNA(s) whose size is approximately 1200 nucleotides. Although the Ya, Yb, and Yc mRNAs are elevated coordinately by phenobarbital and 3-methylcholanthrene, the Ya-Yc mRNAs are induced to a much greater extent compared to the Yb mRNA(s). These data suggest that the mRNAs for each transferase isozyme are regulated independently.     

Preferential over-expression of the class alpha rat Ya2 glutathione S-transferase subunit in livers bearing aflatoxin-induced pre-neoplastic nodules. Comparison of the primary structures of Ya1 and Ya2 with cloned class alpha glutathione S-transferase cDNA sequences.

Normal rat liver expresses Ya (Mr 25,500), Yc (Mr 27,500) and Yk (Mr 25,000) Class Alpha glutathione S-transferase (GST) subunits. The Ya-type subunit can be resolved into two separate polypeptides, designated Ya1 and Ya2, by reverse-phase h.p.l.c. In rat livers that possess aflatoxin B1-induced pre-neoplastic nodules, a marked increase is observed in the expression of Ya1, Ya2, Yc and Yk; of these subunits, Ya2 exhibited the greatest increase in concentration. The Ya1 and Ya2 subunits isolated from nodule-bearing livers were cleaved with CNBr, and the purified peptides were subjected to automated amino-acid-sequence analysis. Differences in the primary structures of the two Ya GST subunits were found at positions 31, 34, 107 and 117. These data demonstrate that Ya1 and Ya2 are distinct polypeptides and are the products of separate genes. The amino acid sequences obtained from Ya1 and Ya2 were compared with the cloned cDNAs pGTB 38 [Pickett, Telakowski-Hopkins, Ding, Argenbright & Lu (1984) J. Biol. Chem. 259, 4112-4115] and pGTR 261 [Lai, Li, Weiss, Reddy & Tu (1984) J. Biol. Chem. 259, 5182-5188], which encode rat Ya-type subunits. From these comparisons it appears probable that Ya1 represents the GST subunit encoded by pGTR 261, whereas Ya2 represents the subunit encoded by pGTB 38. It is likely that the over-expression of Ya1 and Ya2 in nodule-bearing livers is of major significance in the acquired resistance of nodules to aflatoxin B1, since previous work [Coles, Meyer, Ketterer, Stanton & Garner (1985) Carcinogenesis 6, 693-697] has shown that the Ya-type GST subunit has high activity towards aflatoxin B1 8,9-epoxide.     

Ligandin heterogeneity : evidence that the two non-identical subunits are the monomers of two distinct proteins.

Purified ligandin (Y-protein) a 46000-dalton protein, has been shown to consist of two subunit species (mol. wts. 22 000 and 24 000) on discontinuous polyacrylamide gel electrophoresis in sodium dodecyl sulphate. This technique was used to define further the nature of these subunits. The Y sulphobromophthalein-binding fraction of rat hepatic cytosol was shown to contain three major subunit bands designated subunit Ya, subunit Yb and subunit Yc in ascending order of size. Purified ligandin was found to comprise Ya and Yc subunit species, and also gave two bands on isoelectric focusing. The two subunit species in purified ligandin were partially separated by an additional purification step. Antiserum to ligandin reacted mono-specifically with the purified protein, as well as hepatic, renal and small intestinal mucosa cytosol, but gave lines of identity and partial identity with cytosol from testis, ovary and adrenal gland. The Y fraction of testis was found to contain only Yb and Yc species, while all three major bands were found in liver, kidney and small intestinal mucosa. Phenobarbital treatment increased the concentration of Ya and Yb in the liver, but had little effect on Yc. These findings suggest that the Ya and Yc ligandin subunits are the monomers of two proteins: YaYa and YcYc.     

Immunological and sequence interrelationships between multiple human liver and rat glutathione S-transferases

The 13 forms of human liver glutathione S-transferases (GST) (Vander Jagt, D. L., Hunsaker, L. A., Garcia, K. B., and Royer, R. E. (1985) J. Biol. Chem. 260, 11603-11610) are composed of subunits in two electrophoretic mobility groups: Mr = 26,000 (Ha) and Mr = 27,500 (Hb). Preparations purified from the S-hexyl GSH-linked Sepharose 4B affinity column revealed three additional peptides at Mr = 30,800, Mr = 31,200, and Mr = 32,200. Immunoprecipitation of human liver poly(A) RNAs in vitro translation products revealed three classes of GST subunits and related peptides at Mr = 26,000, Mr = 27,500, and Mr = 31,000. The Mr = 26,000 species (Ha) can be precipitated with antisera against a variety of rat liver GSTs containing Ya, Yb, and Yc subunits, whereas the Mr = 27,500 species (Hb) can be immunoprecipitated most efficiently by antiserum against the anionic isozymes as well as a second Yb-containing isozyme (peak V) from the rat liver. The Mr = 31,000 band can be immunoprecipitated by antisera preparations against sheep liver, rat liver, and rat testis isozymes. Human liver GSTs do not have any subunits of the rat liver Yc mobility. Antiserum against the human liver GSTs did not cross-react with the Yc subunits of rat livers or brains in immunoblotting experiments. The human liver GST cDNA clone, pGTH1, selected human liver poly(A) RNAs for the Ha subunit(s) in the hybrid-selected in vitro translation experiments. Southern blot hybridization results revealed cross-hybridization of pGTH1 with the Ya, Yb, and Yc subunit cDNA clones of rat liver GSTs. This sequence homology was substantiated further in that immobilized pGTH1 DNA selected rat liver poly(A) RNAs for the Ya, Yb, and Yc subunits with different efficiency as assayed by in vitro translation and immunoprecipitation. Therefore, we have demonstrated convincingly that sequence homology as well as immunological cross-reactivity exist between GST subunits from several rat tissues and the human liver. Also, the multiple forms of human liver GSTs are most likely encoded by a minimum of three different classes of mRNAs. These results suggest a genetic basis for the subunit heterogeneity of human liver GSTs.     

Purification of glutathione S-transferases from rat liver and walker 256 mammary carcinoma cells by high-performance liquid chromatography and a glutathione affinity column

A novel method for the rapid purification of glutathione S-transferases (GST) from tissue and cell culture samples is reported. A high-performance glutathione affinity column was used and produced results comparable to those obtained with classical agarose affinity columns. Experiments with purified rat liver GST standards resulted in 87% recovery of total activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the affinity-purified GST was identical to the GST standard and revealed three major protein bands, corresponding to the Ya, Yb, and Yc subunits. A fourth protein band (relative molecular mass 25 000), migrating slightly faster than the Ya subunit, was present in both the standard and eluted GST samples. This polypeptide was tentatively identified as the Yk subunit. Successful purification from rat liver and Walker 256 rat carcinoma cell cytosols was also performed. Recovery of total GST enzymatic activity from Walker cell and rat liver cytosol was 49 and 58%, respectively. SDS-PAGE of these samples indicated a high degree of purity. This methodology requires less than 1 h and can be performed using small quantities of tissue. These features make this technique applicable to analysis of a broad range of biological applications including human biopsy material for GST content.     

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.     

Human liver glutathione S-transferases: complete primary sequence of an Ha subunit cDNA

Multiple human liver GSH S-transferases (GST) with overlapping substrate specificities may be essential to their multiple roles in xenobiotics metabolism, drug biotransformation, and protection against peroxidative damage. Human liver GSTs are composed of at least two classes of subunits, Ha (Mr = 26,000) and Hb (Mr = 27,500). Immunological cross-reactivity and nucleic acid hybridization studies revealed a close relationship between the human Ha subunit and rat Ya, Yc subunits and their cDNAs. We have determined the nucleotide sequence of the Ha subunit 1 cDNA, pGTH1. The alignments of its coding sequence with the rat Ya and Yc cDNAs indicate that they are approximately 80% identical base-for-base without any deletion or insertion. Regions of sequence homology (greater than 50%) have also been found between pGTH1 and a corn GST cDNA and rat GST cDNAs of the Yb and Yp subunits. Among the 62 highly conserved amino acid residues of the rat GST supergene family, 56 of them are preserved in the Ha subunit 1 coding sequences. Comparison of amino-acid replacement mutations in these coding sequences revealed that the percentage divergence between the rat Ya and Yc genes is more than that between the Ha and Ya or Ha and Yc genes.     

Multiple Ya subunits of glutathione S-transferase detected by monoclonal antibodies

Monoclonal antibodies to ligandin (YaYa) and glutathione (GSH) S-transferase B (YaYc) were produced by hybridomas derived from the fusion of mouse myeloma cells and spleen cells of mice immunized with the YaYa or YaYc proteins, respectively. Enzyme-linked immunosorbent assay was used to screen for antibody-producing clones. Immunoblotting of the subunits of transferase B, ligandin, and another GSH S-transferase containing Yb subunits showed that the monoclonal antibodies produced by two anti-YaYa subclones recognized the Ya subunits of both ligandin and transferase B, but they did not bind Yc or Yb subunits. It was also revealed that antibodies produced by several anti-YaYc subclones recognized the Yc subunit, but not the Ya subunit of the antigen which was used for the immunization of the mice. However, these monoclonal antibodies did bind the Ya subunit of ligandin. These results indicate that the Ya subunits of GSH S-transferase B and of ligandin do share at least one common determinant. However, these two Ya subunits are structurally distinct as evidenced by their differences in binding by monoclonal anti-YaYc antibodies.     

Isolation of two cholic acid-binding proteins from rat liver cytosol using affinity chromatography

Using an affinity matrix coupled with cholic acid, two proteins that recognise bile acids were isolated from rat liver cytosol. One protein of molecular weight 68 000 was immunologically identical to rat albumin. The other protein was of molecular weight 46 000. On discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis the 46 000 molecular weight protein dissociated to a single band with an RF value identical to the Yb subunit of the bromosulphophthalein-binding fraction (Y-fraction) of whole liver cytosol. The monomers of purified ligandin under these conditions resolved into two bands which corresponded to the Ya and Yc subunits of liver cytosol Y-fraction. Anti-serum to the purified ligandin reacted monospecifically with purified ligandin and whole liver cytosol, but did not cross-react with the Yb dimer eluted from the affinity column. The Yb dimer was shown to possess glutathione-S-transferase activity with a substrate specificity distinct from ligandin but similar to glutathione-S-transferase C. Cholic acid inhibited the catalytic activity of the transferase.     

Cloning and sequence analysis of a cDNA for a rat liver glutathione S-transferase Yb subunit.

We have isolated a Yb-subunit cDNA clone from a GSH S-transferase (GST) cDNA library made from rat liver polysomal poly(A) RNAs. Sequence analysis of one of these cDNA, pGTR200, revealed an open reading frame of 218 amino acids of Mr = 25,915. The deduced sequence is in agreement with the 19 NH2-terminal residues for GST-A. The sequence of pGTR200 differs from another Yb cDNA, pGTA/C44 by four nucleotides and two amino acids in the coding region, thus revealing sequence microheterogeneity. The cDNA insert in pGTR200 also contains 36 nucleotides in the 5' noncoding region and a complete 3' noncoding region. The Yb subunit cDNA shares very limited homology with those of the Ya or Yc cDNAs, but has relatively higher sequence homology to the placental subunit Yp clone pGP5. The mRNA of pGTR200 is not expressed abundantly in rat hearts and seminal vesicles. Therefore, the GST subunit sequence of pGTR200 probably represents a basic Yb subunit. Genomic DNA hybridization patterns showed a complexity consistent with having a multigene family for Yb subunits. Comparison of the amino acid sequences of the Ya, Yb, Yc, and Yp subunits revealed significant conservation of amino acids (approximately 29%) throughout the coding sequences. These results indicate that the rat GSTs are products of at least four different genes that may constitute a supergene family.     

Irreversible inhibition of hepatic glutathione S-transferase by ciprofibrate, a peroxisome proliferator

Ciprofibrate (2-[4-(2,2-dichlorocyclopropyl) phenoxy]2-methyl propionic acid) which is a hypolipidemic agent and has been shown to cause peroxisome proliferation, non-competitively inhibits glutathione S-transferase activity of rat liver, both in vivo and in vitro. Among all the glutathione S-transferases of rat liver, ligandin is maximally inhibited by ciprofibrate. Studies with the purified glutathione S-transferases of rat liver indicate that the affinities of different subunits of liver enzymes for ciprofibrate are in the order Ya greater than Yb, Yb' greater than Yc.