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.     

Covalent labeling of the nonsubstrate ligand-binding site of glutathione S-transferases with bilirubin-Woodward's reagent K

The dimeric enzyme glutathione S-transferase B is composed of two dissimilar subunits, referred to as Ya and Yc. Transferase YaYc and the YaYa homodimer were purified from rat liver cytosol. An enol ester derivative of bilirubin (bilirubin-Woodward's reagent K) was prepared and used to label covalently the nonsubstrate ligand-binding site on these two proteins. There was a linear relationship between the amount of bilirubin-Woodward's reagent K added to the reaction mixture and the amount of labeling achieved up to a ratio of 2:1 (bilirubin-Woodward's reagent K: protein-YaYc). A maximum of 0.87 mol of label bound per mol of transferase YaYc. At higher molar ratios, the label appeared to also be binding at a second site on the enzyme. The label blocked the nonsubstrate ligand-binding site of the two transferases but not the catalytic site. The divalent reagent was shown to label equally the Ya and Yc subunits of transferase YaYc, suggesting that the single high affinity bilirubin-binding site present on this protein is formed by an interaction between the subunits rather than residing on a specific subunit. At low ratios of label to protein, bilirubin-Woodward's reagent K appears to label specifically the nonsubstrate ligand-binding site of two forms of glutathione S-transferase, and use of this label should allow for the localization of the nonsubstrate ligand-binding site in the primary amino acid sequence of the Ya and Yc subunits.     

Preferential binding of steroids by anionic forms of rat glutathione S-transferase

Rat liver glutathione S-transferases with isoelectric points near 6.7 were resolved from more basic forms of the protein. This anionic fraction represented about 30% of the total activity in liver with 1-chloro-2,4-dinitrobenzene and was the preponderant form utilizing trans-4-phenyl-3-butene-2-one as a substrate. The anionic transferases are dimeric proteins composed of two subunits designated as Yb and were distinguished from the cationic transferases on the basis of structural, immunological, and binding properties. Amino acid compositions and immunological properties of the anionic protein were similar to those of glutathione S-transferases A and C. The anionic forms had substantially less ordered secondary structure than cationic forms composed of subunits Ya and Yc. Stoichiometric ratios of two high affinity binding sites per dimer, also differentiated between the anionic and all of the cationic transferases which bind only a single mole of ligand. Affinity matrices composed of corticosterone or cholate, and circular dichroism methods, were used to demonstrate selective binding of steroids and bile acids to the anionic glutathione S-transferases. Glucocorticoids and progestins were shown to bind with high affinity whereas estrogens were bound at distinct lower affinity sites. In contrast to the cationic transferases, glutathione had no effect on binding of the steroids to the anionic forms, which suggested that these proteins have the capacity to bind these substances even in a milieu with high concentrations of glutathione.     

Localization of a portion of the active site of two rat liver glutathione S-transferases using a photoaffinity label

The glutathione S-transferases are a family of dimeric enzymes that catalyze the reaction between GSH and a variety of electrophiles. Two closely related isozymes, referred to as YaYa and YcYc, were purified from rat liver. A radiolabeled azido derivative of glutathione (S-(p-azidophenacyl)[3H]glutathione) was prepared and used to label covalently the active site of the above two glutathione S-transferases. The noncovalently bound affinity label was a competitive inhibitor of glutathione S-transferase YaYa toward both 1-chloro-2,4-dinitrobenzene and GSH. The covalently labeled enzymes no longer bound to a GSH-affinity column, and covalent labeling was reduced in the presence of GSH and S-(dinitrophenyl)glutathione. These results suggest that the affinity label was binding at the active site. The covalently labeled enzymes were digested with trypsin, and the labeled peptides were purified by HPLC and then sequenced. A single-labeled peptide was identified in the tryptic digest of the YaYa isozyme, whereas two labeled peptides were present in the tryptic digest of YcYc. The Ya peptide sequence was identical with the published deduced sequence of amino acids between residues 212 and 218 and the sequences of the two peptides purified from Yc were identical with the deduced sequence of amino acids between 91 and 110 and 206 and 218. Hence, the Ya peptide and the smaller peptide purified from Yc came from the same region of the Ya and Yc subunits. This common region and a second region of the Yc subunit appear to form a portion of the active site of these two forms of glutathione S-transferase.     

Identity of ligandin in rat testis and liver.

1. One of the main problems in the field of multifunctional proteins such as ligandin is the possibility that multiple forms and isoproteins may exist. Because liver ligandin [GSH (reduced glutathione) S-transferase B] consists of equal amounts of Ya (22 000 Da) and Yc (25 000 Da) subunits, and testis ligandin, prepared by the standard technique of anion-exchange and molecular-exclusion chromatography, contains more Yc subunit than Ya, it has been claimed that testis and liver ligandin are different entities. 2. We purified testis ligandin by immunoaffinity chromatography and have obtained a product identical with liver ligandin (Yc = Ya). This suggests that the differences previously described may be due to contamination of testis ligandin by a closely related species. In fact sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of testis GSH S-transferases separated by CM-cellulose chromatography showed that GSH S-transferase AA, present in large amounts, migrated in the same region as Yc subunit. 3. Testis ligandin prepared by the standard technique was similar to that reported [Bhargava, Ohmi, Listowsky & Arias (1980) J. Biol. Chem. 255, 724-727] and contained more Yc subunit than Ya. CM-cellulose chromatography of this 'pure' preparation revealed significant amounts of GSH S-transferase AA migrating as Yc subunit, in addition to ligandin consisting of equal amounts of Ya and Yc subunits. 4. Our studies show that testis ligandin is identical with liver ligandin. Previously described differences are due to a contaminant identified as GSH S-transferase AA.     

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.     

Studies of the relationship between the catalytic activity and binding of non-substrate ligands by the glutathione S-transferases.

The dimeric enzyme glutathione S-transferase B is composed of two dissimilar subunits, referred to as Ya and Yc. Transferase B (YaYc) and two other transferases that are homodimers of the individual Ya and Yc subunits were purified from rat liver. Inhibition of these three enzymes by Indocyanine Green, biliverdin and several bile acids was investigated at different values of pH (range 6.0-8.0). Indocyanine Green, biliverdin and chenodeoxycholate were found to be effective inhibitors of transferases YaYc and YcYc at low (pH 6.0) but not high (pH 8.0) values of pH. Between these extremes of pH intermediate degrees of inhibition were observed. Cholate and taurochenodeoxycholate, however, were ineffective inhibitors of transferase YcYc at all values of pH. The observed differences in bile acids appeared to be due, in part, to differences in their state of ionization. In contrast with the above results, transferase YaYa was inhibited by at least 80% by the non-substrate ligands at all values of pH. These effects of pH on the three transferases could not be accounted for by pH-induced changes in the enzyme's affinity for the inhibitor. Thus those glutathione S-transferases that contain the Yc subunit are able to act simultaneously as both enzymes and binding proteins. In addition to enzyme structure, the state of ionization of the non-substrate ligands may also influence whether the transferases can perform both functions simultaneously.     

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.     

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.     

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].     

Chromosomal assignments of genes for rat glutathione S-transferase Ya (GSTA1) and Yc subunits (GSTA2).

Chromosomal assignments of genes for rat glutathione S-transferase Ya (GSTA1) and Yc subunits (GSTA2) were performed by Southern blot analyses of somatic cell hybrid DNAs.GSTA1 and GSTA2 were assigned to rat chromosomes 8 and 9, respectively.     

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 glutathione S-transferase. Cloning of double-stranded cDNA and induction of its mRNA

Messenger RNA extracted from the livers of normal, phenobarbital-treated, and trans-stilbene oxide-treated rats was translated in a mRNA-dependent protein-synthesizing system. Immunoprecipitation of the translation products by antibodies against the Ya and Yc subunits of glutathione S-transferase detected two polypeptides of molecular weights 23,500 and 25,000. Subsequently, a clone containing glutathione S-transferase sequences was identified from a rat liver double-stranded cDNA library that had been prepared by homopolymeric tailing and cloning into the Pst I site of pBR322. Confirmation of the identity of the clone was obtained by recloning the 550-bp insert DNA into the phage vector M13 and utilizing the single strand recombinant phage DNA in specific hybrid selection of mRNA followed by translation and immunoprecipitation with antibodies to the Ya and Yc subunits. This recombinant phage, M13GST94, was also utilized in a new technique to synthesize 32P-labeled cDNA specific to the glutathione S-transferase insert DNA that was used subsequently in RNA excess solution hybridization to determine the relative concentration of glutathione S-transferase mRNA. Phenobarbital treatment resulted in a 3.2-fold increase in glutathione S-transferase mRNA over levels found in control rats, while trans-stilbene oxide increased glutathione S-transferase mRNA levels 5.7-fold. The DNA sequence of the clone was determined and utilized to propose a partial amino acid sequence.     

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.     

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.     

Cloning and the nucleotide sequence of rat glutathione S-transferase P cDNA.

A cDNA library prepared from poly(A)+ RNA of 2-acetylaminofluorene (AAF) induced rat hepatocellular carcinoma was screened by synthetic DNA probes deduced from a partial amino acid sequence of glutathione S-transferase P subunit that had been isolated from the tumor by two-dimensional gel electrophoresis. One of the four clones analyzed contained an mRNA region encoding the total amino acid sequence of this enzyme subunit and the complete 3'-noncoding region. The nucleotide sequence indicates that this enzyme subunit has 209 amino acids (calculated Mr=23,307) distinct from other glutathione S-transferase subunits such as Ya and Yc. Comparison of the amino acid sequences between these proteins indicates that glutathione S-transferase P subunit gene has been evolved from the ancestral gene at an earlier stage than the separation of Ya and Yc and that there are at least three domains having a considerable homology with each other in these enzymes. The very large increase of this mRNA in chemically induced hepatocellular carcinoma suggests a characteristic derepression of this gene during hepatocarcinogenesis.     

Protection of glutathione S-transferase from bilirubin inhibition

Inhibition of the enzyme activity of glutathione S-transferase (GST) by a physiological concentration of bilirubin was studied using various substrates. When rat liver cytosol was used as an unfractionated GST, its GSH-conjugation activity toward 1-chloro-2,4-dinitrobenzene was decreased to one-half by bilirubin, while the activity toward 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, or 1,2-epoxy-(p-nitrophenoxy)propane and also the non-selenium dependent GSH-peroxidase activity toward cumene hydroperoxide (CHPx activity) were hardly affected under the same conditions. In contrast, bilirubin inhibited each of the purified GST isozymes and no remarkable difference in bilirubin inhibition was observed with any of the substrates tested. From the chromatographic analysis of the cytosol incubated with [3H]bilirubin, it was found that a major part of the added bilirubin binds to subunit 1 (Ya) of GST isozyme, leaving not only the conjugation activity derived from 3-4 type GST but also the CHPx activity of subunit 2 (Yc) quantitatively intact. The bilirubin inhibition of both the conjugation activity of GST 3-4 and the CHPx activity of GST 2-2 was prevented almost completely by addition of a 3-fold molar excess of GST 1-1. From these results, it was assumed that the enzyme activities of both 3-4 type GSTs and subunit 2 (Yc) were protected from the inhibitory action of bilirubin by the scavenger effect of subunit 1 (Ya).     

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.     

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.     

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing. Bacterial clones harboring inserts complementary to glutathione S-transferase mRNA were identified by colony hybridization using a [32P]cDNA probe reverse transcribed from poly(A+)-RNA enriched significantly in glutathione S-transferase B mRNA and by hybrid-select translation. Two recombinant clones, pGTB6 and pGTB15 hybrid-selected the mRNAs specific for the Ya and Yc subunits, indicating these two mRNAs share significant sequence homology. Radiolabeled pGTB6 was utilized in RNA gel-blot experiments to determine that the size of glutathione S-transferase B mRNA is 980 nucleotides and the degree of induction of the mRNA in response to 3-methylcholanthrene administration is threefold.