The nucleotide sequence of a rat liver glutathione S-transferase subunit cDNA clone

We have determined the nucleotide sequence of a cloned cDNA derived from liver poly(A) RNA of pentobarbital-treated rats encoding a glutathione S-transferase subunit. This cDNA clone pGTR261 contains one open reading frame of 222 amino acids, a complete 3' noncoding region, and 63 nucleotides in the 5' noncoding region. The cloned DNA hybridizes to rat poly(A) RNA in a tissue-specific fashion, with strong signals to liver and kidney poly(A) RNA(s) of approximately 1100 and approximately 1400 nucleotides in size but little or no hybridization to poly(A) RNAs from heart, lung, seminal vesicles, spleen, or testis under stringent conditions. Our sequence covers the cDNA sequence of pGST94 which contains a partial coding sequence for a liver glutathione S-transferase subunit of Ya size. Comparison of sequences with our earlier clone pGTR112 suggests that there are at least two mRNA species coding for two different subunits of the Ya (Mr = 25,600) subunit family with very limited amino acid substitutions mainly of conserved polarity. The divergent 3' noncoding sequences should be useful molecular probes in differentiating these two different but otherwise very similar subunits in induction and genomic structure analyses. Our results suggest that tissue-specific expression of the glutathione S-transferase subunits represented by the sequences of pGTR261 and pGTR112 may occur at or prior to the level of RNA processing.     

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.     

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.     

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.     

Selective induction of glutathione S-transferase D in rat testis by phenobarbital

Testis cytosol is shown to contain the Yb2Yb2 -homodimer glutathione S-transferase D in addition to the previously described glutathione S-transferases A ( Yb1Yb1 ) and C ( Yb1Yb2 ). Treatment of rats with phenobarbital induces the level of glutathione S-transferase D in testis with no increase in the activities of glutathione S-transferases A and C. This result indicates a specific induction of the Yb2 subunit in testis, in contrast with the situation in rat liver, where phenobarbital specifically induces the Yb1 subunit.     

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.     

Human glutathione S-transferases. Characterization of the anionic forms from lung and placenta.

Anionic glutathione S-transferases were purified from human lung and placenta. Chemical and immunochemical characterization, including polyacrylamide-gel electrophoresis, gave strong evidence that the anionic lung and placental enzymes are chemically similar, if not identical, proteins. The electrophoretic mobilities of both proteins were identical in conventional alkaline gels as well as in gels containing sodium dodecyl sulphate. Gel filtration of the intact active enzyme established an Mr value of 45000; however, with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under dissociating conditions a subunit Mr of 22500 was obtained. Amino acid sequence analysis of the N-terminal region of the placental enzyme revealed a single polypeptide sequence identical with that of lung. Results obtained from immunoelectrophoresis, immunotitration, double immunodiffusion and rocket immunoelectrophoresis also indicated the anionic lung and placental enzymes to be closely similar. The chemical similarity of these two proteins was further supported by protein compositional analysis and fragment analysis after chemical hydrolysis. Immunochemical comparison of the anionic lung and placental enzymes with human liver glutathione S-transferases revealed cross-reactivity with the anionic omega enzyme, but no cross-reactivity was detectable with the cationic enzymes. Comparison of the N-terminal region of the human anionic enzyme with reported sequences of rat liver glutathione S-transferases gave strong evidence of chemical similarity, indicating that these enzymes are evolutionarily related. However, computer analysis of the 30-residue N-terminal sequence did not show any significant chemical similarity to any other reported protein sequence, pointing to the fact that the glutathione S-transferases represent a unique class of proteins.     

Suppression of glutathione S-transferases in rat seminal vesicles or pituitary glands

We have studied the tissue-specific expression of GSH S-transferases in rat seminal vesicles and pituitary glands by in vitro translation and immunoprecipitation. The major GSH S-transferase subunit expressed in rat seminal vesicles belongs to the Yb mobility class whose expression diminishes when the rats are treated with pentobarbital. The pattern of GSH S-transferase expression in the pituitary gland is very similar to that of the rat brain with Yb size subunit(s) predominant. The Y beta size subunit is also expressed together with the Yc and Y delta subunits. The expression of GSH S-transferases was drastically reduced in pituitary gland poly(A) RNAs from diethylstilbestrol-treated, ovariectomized female rats. Xenobiotics such as phenobarbital, 3-methylcholanthrene, and trans-stilbene oxide induce rat liver GSH S-transferase activities, especially the Ya- and Yb-subunit containing isozymes. Induction of GSH S-transferases by a combination of the three xenobiotics is neither additive nor synergistic, however. Our results clearly demonstrate that GSH S-transferase expression in seminal vesicles and pituitary glands can be suppressed by phenobarbital and diethylstilbestrol, respectively. Our findings suggest that different GSH S-transferase isozymes respond differently to various xenobiotics. Both induction and suppression occur in rats treated with xenobiotics. This notion helps to explain the lack of additive or synergistic induction in rats treated with more than one xenobiotic.     

Tissue distribution and subunit structures of the multiple forms of glutathione S-transferase in the rat.

A study of the subunit structures of the multiple forms of glutathione S-transferase in rat kidney, testis, lung and spleen is shown to be consistent with a proteolytic model for the generation of the multiple forms.     

Comparative effect of the induction of the subunits of rat liver glutathione S-transferases by butylated hydroxytoluene

When butylated hydroxytoluene (BHT) was administered to rats, the smallest subunit Ya (Mr 22,000) of rat liver GSH S-transferases was found to undergo maximum induction. It is suggested that the differential induction of GSH S-transferase activities by BHT towards different substrates may be due to the differences in the induction of the constituent subunits of GSH S-transferases.     

Subunit composition of rat liver glutathione S-transferases

The plasmid pGTR112 contains partial coding sequences for one of the rat liver glutathione S-transferase subunits. We have used immobilized pGTR112 DNA to select for complementary and homologous liver poly(A)-RNAs under conditions of increasing stringency for hybridization. Each fraction of selected poly(A)-RNAs was assayed by in vitro translation followed by immunoprecipitation. A total of four distinct polypeptides precipitated by antiserum against rat liver glutathione S-transferases were resolved by NaDodSO₄ polyacrylamide gel electrophoresis. They are separated into two pairs according to the sequence homology of their poly(A)-RNAs with the pGTR112 DNA. Purified rat liver glutathione S-transferases can be resolved on gradient NaDodSO₄ polyacrylamide gels into four polypeptides. There should be ten isozymes of different binary combinations from four distinct subunits for the rat liver glutathione S-transferases.     

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.     

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.     

A new class of rat glutathione S-transferase Yrs-Yrs inactivating reactive sulfate esters as metabolites of carcinogenic arylmethanols

A glutathione (GSH) S-transferase (GST), catalyzing the inactivation of reactive sulfate esters as metabolites of carcinogenic arylmethanols, was isolated from the male Sprague-Dawley rat liver cytosol and purified to homogeneity in 12% yield with a purification factor of 901-fold. The purified GST was a homo-dimeric enzyme protein with subunit Mr 26,000 and pI 7.9 and designated as Yrs-Yrs because of its enzyme activity toward "reactive sulfate esters." GST Yrs-Yrs could neither be retained on the S-hexylglutathione gel column nor showed any activity toward 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and 1,2-epoxy-3-(4'-nitrophenoxy)propane. 1-Chloro-2,4-dinitro-benzene was a very poor substrate for this GST. 1-Menaphthyl sulfate was the best substrate for GST Yrs-Yrs among the examined mutagenic arylmethyl sulfates. The enzyme had higher activities toward ethacrynic acid and cumene hydroperoxide. N-terminal amino acid sequence of subunit Yrs, analyzed up to the 25th amino acid, had no homology with any of the known class alpha, mu, and pi enzymes of the Sprague-Dawley rat. Anti-Yrs-IgG raised against GST Yrs-Yrs showed no cross-reactivity with any of subunits Ya, Yc, Yb1, Yb2, and Yp. Anti-IgGs raised against Ya, Yc, Yb1, Yb2, and Yp also showed no cross-reactivity with GST Yrs-Yrs. The purified enzyme proved to differ evidently from the 12 known cytosolic GSTs in various tissues of the rat in all respects. Immunoblot analysis of various tissue cytosols of the male rat indicated that apparent concentrations of the GST Yrs-Yrs protein were in order of liver greater than testis greater than adrenal greater than kidney greater than lung greater than brain greater than skeletal muscle congruent to heart congruent to small intestine congruent to spleen congruent to skin congruent to 0.     

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)     

Glutathione S-transferase distribution and concentration in human organs

The concentration of basic, near-neutral and acid GSH S-transferase was measured in 18 organs from each of 9 male human subjects using radial immunodiffusion. Basic transferases were detectable in all tissues studied. Highest concentrations were found in liver, testis, kidney, adrenal and jejunum while low levels were found in bladder, muscle and thyroid. The concentration in liver was 230 times higher than that in thyroid. Near-neutral GSH S-transferase were absent in all tissues in 5 of the 9 individuals studied. When present they were widely distributed, highest concentrations being found in liver, testis, muscle, adrenal and brain and lowest levels in thyroid, lung, duodenum, stomach, heart and kidney. Acid GSH S-transferases were present in every individual studied although they were undetectable in the liver of a single subject. Highest concentrations were present in colon, jejunum, ileum, bladder, spleen and lung while low concentrations were found in liver. Our study provides conclusive evidence of marked inter-individual and inter-organ variation of the three groups of human GSH S-transferase.     

Cloning and expression in Escherichia coli of cDNA for arginase of rat liver

A cDNA expression library constructed in a plasmid pUC8 from poly(A)+ RNA of rat liver was screened immunologically, using an antibody against arginase of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.35 kilobase pairs in length. In the bacterial clone, we detected a specific protein of Mr = about 43,000 that is slightly larger than the purified arginase (Mr = about 40,000) and a high activity of arginase was expressed. The arginase mRNA species of about 1600 bases long was detected in the liver, but not in the small intestine, kidney, spleen and heart of the rats.     

Glutathione S-transferases in human prostate

A number of human prostatic tissue biopsies have been analyzed for glutathione S-transferase activity, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Samples from nine patients (age range 61-90) with benign prostatic hypertrophy who had received no prior chemotherapy had a mean glutathione S-transferase activity of 137 +/- 44 nmol/min per mg with a range of 97-237. A qualitative comparison of the glutathione S-transferase of normal prostate and benign prostatic hypertrophy samples was carried out. Approximately 260-fold purification was achieved using glutathione-Sepharose affinity chromatography, with glutathione S-transferase accounting for approximately 0.19-0.33% of the total protein. Substrate specificity determinations suggested similar, but not identical, glutathione S-transferase subunits in normal prostate and benign prostatic hypertrophy. One- and two-dimensional electrophoresis (isoelectric focusing and 12.5% SDS-polyacrylamide gel electrophoresis) identified at least seven stained polypeptides in the purified glutathione S-transferase preparations. These ranged in Mr from approximately 24,000 to 28,500 and in pI from near neutral to basic. Western blot analysis using polyclonal antibodies raised against rat liver glutathione S-transferase suggested crossreactivity with five of the human isoenzymes in both normal prostate and benign prostatic hypertrophy. One of the glutathione S-transferases, present in both normal prostate and benign prostatic hypertrophy, had an Mr of approx. 24,000 and a near-neutral pI and crossreacted immunologically with a polyclonal antibody raised against human placental glutathione S-transferase (Yf, subunit 7 or pi). These data suggest that four glutathione S-transferases are expressed in human prostate, with subunits from each of the major classes alpha, mu and pi. These are characterized as Ya, Yb, Yb' and Yf (analogous alternative nomenclature subunits 1, 3, 4 and 7).