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

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

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

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

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 and partial characterization of the glutathione S-transferases in carp liver, and their interaction with 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone

The hepatic glutathione S-transferase (GST) activity in the cytosol of the freshwater fish carp (Cyprinus carpio) was enriched by glutathione affinity chromatography. The anionic (GST A1-A3) and cationic (GST C1-C3) isoenzymes were then separated in two chromatofocusing steps. SDS electrophoresis showed GST C1 to be a heterodimer with subunits of Mr 25,000 and 28,000, and all other isoenzymes to be homodimers with subunits of Mr 25,400. They were partially characterized by different biochemical parameters. The water pollutants 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone inhibited all carp GST isoenzymes, following the same kinetic inhibition patterns as for rat liver GST. It is concluded that hepatic carp GST can play an important role in the detoxication of aquatic pollutants.     

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.     

Comparative study of the purification and characterization of the cytosolic glutathione S-transferases from two salmonid species: Atlantic salmon (Salmo salar) and brown trout (Salmo trutta)

In the present report, an efficient method for isolating multiple cytosolic forms of glutathione S-transferases from liver and kidney cytosolic samples of two salmonid species (brown trout and Atlantic salmon) is described, and some of the multiple properties of these enzymes are presented. Glutathione S-transferases were partially purified by low-pressure affinity chromatography on a column with glutathione coupled to agarose, which retained an average of 89.47% of the total activity. The GST activity was appropriated towards CDNB and ETHA as substrates. The application of an HPLC system associated to elestrospray ionization mass spectrometry allowed the identification of five GST cytosolic isoforms, corresponding to subunits with M(r) between 23,700 and 26,900 Da being the main form, with retention time of 17 min, a pi-class-related GST isoenzyme.     

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.     

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.     

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.     

The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene

In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.     

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.     

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.     

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.     

Subunit composition of glutathione S-transferases of rat lung

Rat lung glutathione S-transferases (GST) III (pI 6.8) and IV (pI 6.0) have two immunologically and kinetically distinct Yb types of subunits and these subunits are immunologically similar to the corresponding subunits of liver GST. It is demonstrated that GST III (pI 6.8) and IV (pI 6.0) of rat lung are heterodimers of Ya and Yb type of subunits, a combination not observed among liver GST. Unlike the Yb type subunits of rat liver GST, the Yb type subunits of rat lung GST hybridize with the Ya type subunits in vitro.     

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.     

Purification and partial characterization of seven glutathione S-transferase isoforms from the clam Ruditapes decussatus.

This paper deals with the purification and the partial characterization of glutathione S-transferase (GST) isoforms from the clam Ruditapes decussatus. For the first step of purification, two affinity columns, reduced glutathione (GSH)-agarose and S-hexyl GSH-agarose, were mounted in series. Four affinity fractions were thus recovered. Further purification was performed using anion exchange chromatography. Seven fractions, which present a GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP-HPLC. Seven distinct GST isoforms were purified, six of them were homodimers, the last one was a heterodimer consisting of the subunits 3 and 6. Kinetic parameters were studied. Results showed that isoforms have distinct affinity and Vmax for GSH and CDNB as substrates. The catalytic activity of the heterodimer isoform appeared to be a combination of the ability of each subunit. The immunological properties of each purified isoform were investigated using three antisera anti-pi, anti-mu and anti-alpha mammalian GST classes. Three isoforms (3-3, 6-6 and 3-6) seem to be closely related to the pi-class GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera to pi and alpha classes and the isoform 5-5 cross-reacted with the antisera to mu and pi classes. Subunit 4 was recognized by the three antisera used, and its N-terminal amino acid analysis showed high identity (53%) with a conserved sequence of an alpha/m micro /pi GST from Fasciola hepatica.     

Apolipoproteins and the association of egg yolk proteins with plasma high density lipoproteins after ovulation and follicular atresia in the rainbow trout (Salmo gairdneri)

The apolipoproteins of trout plasma lipoproteins have been characterized by sodium dodecyl sulfate-glycerol polyacrylamide gel electrophoresis. The high density lipoproteins (HDL) (1.085 less than d less than 1.21 g/ml) contain four apolipoproteins, two major species with Mr 25,000 (apoA-I-like) and Mr 13,000 (apoA-II-like) and two minor species (Mr 55,000 and 40,500). The very low density (d less than 1.015 g/ml) and low density lipoproteins (1.015 less than d less than 1.085 g/ml) contain two high Mr apolipoproteins (apoB-like) with Mr 260,000 and 240,000 (the smaller is the preponderant species in low density lipoproteins), as well as a third apolipoprotein with Mr 76,000. Type A apolipoproteins are present in the very low density lipoproteins, as are a group of apolipoproteins with Mr 9,000-11,000 (apoC-like). Egg yolk proteins appear in the plasma of females about 30 days after natural ovulation or after that induced by salmon gonadotropin and during massive intraovarian atresias, either spontaneous or induced by 17 alpha,20 beta-dihydroxy-4-pregnen-3-one. Two egg yolk proteins intimately associated with HDL have been identified. They may account for as much as 35% of total plasma proteins. Lipovitellin (Mr 112,000) is composed of two subunits in a 1:1 molar ratio (lipovitellin 1 with Mr 92,000 and lipovitellin 2 with Mr 20,000) and is present as a dimer with another yolk protein (Mr 10,000). These results show that resorption of the yolk during follicular atresia in an oviparous vertebrate is correlated with the presence of egg yolk proteins combined with HDL in the plasma.     

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.     

Sex differences in the subunits of glutathione-S-transferase isoenzyme from rat and human kidney

Glutathione-S-transferase (GST) isoenzymes were purified from cytosolic preparations from kidneys of male and female rats and kidney cortical specimens from 2 male and 1 female human subjects. GST isoenzyme expression was analyzed by SDS-PAGE, measurement of catalytic activities with specific substrates and determination of their subunits by ELISA and Western blotting using specific antibodies. GST from female rat kidneys showed a preponderance of subunits 3 and 4; levels of these isoenzymes were 3-4 times greater in females than in males. Levels of subunits 1 and 2 were 1.5-2 times greater in the male rat kidneys. Additional minor bands at 24 and 22 kD were observed in GST preparations from both male and female rat kidneys while a band at 25.3 kD was observed only in the male rat kidney. These bands did not react with antibodies to GST 1-1, GST 2-2 or GST 3-4. Both male and female human kidney samples contained GST isoenzymes comparable to the near-neutral (25-5 kD) and basic forms (25 kD) of GSTs found in human liver. In addition a 28-kD band was present in GST preparations from both male and female human kidneys. Additional bands at 29 and 25.2 kD were present only in male human kidneys. Both the kidney cytosol and the total GSTs prepared from female rats shared 2- to 4-fold greater activity with 1,2-dichloro-4-nitrobenzene, ethacrynic acid and trans-4-phenyl-3-buten-2-one than those from males. The measurement of specific subunit amounts by ELISA were in agreement with these results.(ABSTRACT TRUNCATED AT 250 WORDS)