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.     

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.     

Tissue-specific expression of the rat glutathione S-transferases

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

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.     

On the multiplicity of rat liver glutathione S-transferases

Rat liver glutathione S-transferases have been purified to apparent electrophoretic homogeneity by S-hexylglutathione-linked Sepharose 6B affinity chromatography and CM-cellulose column chromatography. At least 11 transferase activity peaks can be resolved including five Yb size homodimeric isozymes, two Yc size homodimeric isozymes, one Ya homodimeric isozyme, one Y alpha homodimeric isozyme, and two Ya-Yc heterodimeric isozymes. Distribution of the GSH peroxidase activity among the CM-cellulose column fractions suggests the existence of further multiplicity in this isozyme family. Substrate specificity patterns of the Yb subunit isozymes revealed a possibility that each of the five Yb-containing isozymes is composed of a different homodimeric Yb size subunit composition. Our findings on the increasing multiplicity of glutathione S-transferase isozymes are consistent with the notion that multiple isozymes of overlapping substrate specificities are required to detoxify a multitude of xenobiotics in addition to serving other important physiological functions.     

Comparison of renal and hepatic glutathione S-transferases in the rat.

Renal and hepatic GSH (reduced glutathione) S-transferase were compared with respect to substrate and inhibitory kinetics and hormonal influences in vivo. An example of each of five classes of substrates (aryl, aralkyl, epoxide, alkyl and alkene) was used. In the gel filtration of renal or hepatic cytosol, an identical elution volume was found for all the transferase activities. Close correspondence in Km values was found for aryl, epoxide- and alkyl-transferase activities, with only the aralkyl activity significantly lower in kidney. Probenecid and p-aminohippurate were competitive inhibitors of renal aryl-, aralkyl-, epoxide- and alkyl-transferase activities and inhibited renal alkene activity. Close correspondence in Ki values for inhibition by probenecid of these activities in kidney and liver was found. In addition, furosemide was a potent competitive inhibitor of renal alkyl-transferase activity. Hypophysectomy resulted in significant increases in aryl-, araklyl-, and expoxide-transferase activities in liver and kidney. The hypophysectomy-induced increases in renal aryl- and aralkyl-transferase activities (approx. 100%) were more than twofold greater than increases in hepatic activities (approx. 40%). Administration of thyroxine prevented the hypophysectomy-induced increase in aryltransferase activity in both kidney and liver. The renal GSH S-transferases, in view of similarities to the hepatic activities, may play a role as cytoplasmic organic-anion receptors, as previously proposed for the hepatic enzymes.     

Subunit structure of human and rat glutathione S-transferases

In rat tissues different forms of glutathione (GSH) S-transferases represent various dimeric combinations of at least four different classes of subunits categorized on the basis of their Mr values as seen on polyacrylamide gels. These subunit types represent heterogeneous populations and the actual number of subunits in rat GSH S-transferases may be far more than is known at present. Human GSH S-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits which can be classified into three types, A (Mr 26,500), B (Mr 24,500) and C (Mr 22,500). There is evidence for considerable charge heterogeneity in each of these subunit types.     

Cholic acid binding by glutathione S-transferases from rat liver cytosol.

Cholic acid-binding activity in cytosol from rat livers appears to be mainly associated with enzymes having glutathione S-transferase activity; at least four of the enzymes in this group can bind the bile acid. Examination of the subunit compositions of different glutathione S-transferases indicated that cholic acid binding and the ability to conjugate reduced glutathione with 1,2-dichloro-4-nitrobenzene may be ascribed to different subunits.     

Acidic and basic forms of glutathione S-transferases from human placenta and comparison with human kidney glutathione S-transferase

Glutathione S-transferase (GSH-transferase) was purified from human placenta and kidney by affinity chromatography on S-glutathione-carbamidomethyl-epsilon-aminolysyl-Sepharose CL 4B and gel filtration chromatography on Sephades G-75. Electrophoretically pure enzyme with the specific activities of 50.7 and 55.9 U/mg, respectively, were obtained. In addition to the known acidic isoenzyme from human placenta (isoelectric point, pI, 4.5), we describe here for the first time the presence of 6 basic forms with pI values between 8.0 and 9.0. The kidney GSH-transferase contained 2 acidic forms with isoelectric points at 4.6 and 4.65, and 6 basic forms with pI values between 8.7 and 9.4. The basic and acidic isoenzymes from placenta were separated by ion exchange chromatography on Sephadex DEAE A-25. The acidic form accounted for 36% of the total GSH-transferase activity from placenta. Antibodies against the kidney enzyme were raised in rabbit. Total cross-reactivity of placental GSH-transferase with antikidney-GSH-transferase antibodies was obtained, suggesting that the kidney and placental enzymes are immunologically closely related.     

A comparison of the glutathione S-transferases of trout and rat liver.

1. Cytosol from trout liver, gills and intestinal caeca has substantial glutathione S-transferase activity. 2. Gel-exclusion and ion-exchange chromatography suggest that trout liver has several glutathione S-transferases with different molecular weights and ionic charges. 3. A component capable of binding lithocholic acid eluted together with glutathione S-transferase activity. Some of the transferase activity did not elute together with binding activity. 4. The enzymic activity from trout liver was less stable at 37 degrees C than that from rat liver. 5. The glutathione S-transferases of fish liver have a similar specific activity to those of rat liver but different molecular properties.     

Purification and characterization of glutathione S-transferases from rat pancreas

Glutathione S-transferases (GSTs) of rat pancreas have been characterized and their interrelationship with fatty acid ethyl ester synthase (FAEES) has been studied. Seven GST isozymes with pI values of 9.2, 8.15, 7.8, 7.0, 6.3, 5.9 and 5.4 have been isolated and designated as rat pancreas GST suffixed by their pI values. Structural, immunological and kinetic properties of these isozymes indicated that GST 9.2 belonged to the alpha class, GST 7.8, 7.0, 6.3 and 5.9 belonged to the mu class, whereas GST 8.15 and 5.4 belong to pi class. The N-terminal sequences and pI values of the mu class isozymes suggested that rat GST subunits 3, 4 and 6 may be expressed in pancreas. N-Terminal sequences of both the pi class isozymes, GST 8.15 and 5.4, were similar to that of GST-P, but there were significant differences in the substrate specificities of these two enzymes. Results of peptide finger print studies also indicated minor structural differences between these two isozymes. None of the GST isozymes of rat pancreas expressed FAEES activity. Rat pancreas had a significant amount of FAEES activity, but it segregated independently during the purification of GST indicating that these two activities are expressed by different proteins and are not related as suggested previously.     

Hybrid formation of rabbit and rat hepatic glutathione S-transferases

1. A reconstitution experiment resulted in the formation of new proteins between limited combinations of rat and rabbit hepatic glutathione S-transferases: AA(subunit composition: YcYc) and R3b(Y3Y3), Lig(YaYa) and R3b(Y3Y3), and A(Yb1Yb1) and R2(Y2Y2). 2. It was demonstrated that the new protein formed between R2 and A had the subunit composition of Y2Yb1, suggesting a hybrid of rabbit (R2) and rat isozyme (A). 3. This hybrid protein showed intermediate spec. acts between those of R2 and A when either 1-chloro-2,4-dinitrobenzene (CDNB) or 1,2-dichloro-4-nitrobenzene (DCNB) was employed as the substrate.     

Characterization of glutathione S-transferases in rat kidney. Alteration of composition by cis-platinum.

We have developed chromatographic and mathematical protocols that allowed the high resolution of glutathione S-transferase (GST) subunits, and the identification of a previously unresolved GST monomer in rat kidney cytosol; the monomer was identified tentatively as subunit 6. Also, an aberrant form of GST 7-7 dimer appeared to be present in the kidney. This development was utilized to illustrate the response of rat kidney GST following cis-platinum treatment in vivo. Rat kidney cytosol was separated into three 'affinity families' of GST activity after elution from a GSH-agarose matrix. The affinity peaks were characterized by quantitative differences in their subunit and dimeric compositions as determined by subsequent chromatography on a cation-exchange matrix and specific activity towards substrates. By use of these criteria, the major GST dimers of affinity peaks were tentatively identified. The major GST dimers in peak I were GST 1-1 and 1-2, in affinity peak II it was GST 2-2, and in peak III they were GST 3-3 and 7-7. GST 3-6 and/or 4-6, which have not been previously resolved in kidney cytosol, were also present in peak II. Alterations in the kidney cytosolic GST composition of male rats were detected subsequent to the administration of cis-platinum (7.0 mg/kg subcutaneously, 6 days). This treatment caused a pronounced alteration in the GST profile, and the pattern of alteration was markedly different from that reported for other chemicals in the kidney or in the liver. In general, the cellular contents of the GSTs of the Alpha and the Mu classes decreased and increased respectively. It is postulated that the decrease in the Alpha class of GSTs by cis-platinum treatment may be related to renal cortical damage and the loss of GSTs in the urine. The increase in the Mu class of GSTs could potentially stem from a lowered serum concentration of testosterone; the latter is a known effect of cis-platinum treatment.     

Interaction of the mycotoxin penicillic acid with glutathione and rat liver glutathione S-transferases

The in vitro interaction of the mycotoxin penicillic acid (PA) with rat liver glutathione S-transferase (GST) was studied using reduced glutathione and 1-chloro-2,4-dinitrobenzene as substrates. The inhibition of the GST activity by PA in crude extracts was dose dependent. Each of the different GST isoenzymes was inhibited, albeit at different degrees. Kinetic studies never revealed competitive inhibition kinetics. The conjugation of PA with GSH occurred spontaneously; it was not enzymatically catalyzed by GST, indicating that an epoxide intermediate is not involved in conjugation. The direct binding of PA to GST provides an additional detoxication mechanism.     

Quantification of human hepatic glutathione S-transferases.

Human hepatic glutathione S-transferase (GST) subunits were characterized and quantified with the aid of a recently developed h.p.l.c. method. In 20 hepatic tissue specimens the absolute amounts of the basic Class Alpha subunits B1 and B2, the near-neutral Class Mu subunits mu and psi and the acidic subunit pi were determined. The average total amount of GST was 37 micrograms/mg of cytosolic protein, with the Class Alpha GST being the predominant class (84% of total GSTs), and pi as the sole representative of the Class Pi GSTs present in the lowest concentration (4% of total GSTs). Large interindividual differences were observed for all subunits, with variations up to 27-fold, depending on the subunit. For the Class Alpha GST-subunits B1 and B2, a biphasic ratio was observed. The genetic polymorphism of the subunits mu and psi was confirmed by h.p.l.c. analysis, and correlated with the enzymic glutathione conjugation of trans-stilbene oxide and with Western blotting of cytosols, using a monoclonal anti-(Class Mu GST) antibody. Of the 20 livers examined, ten contained only mu, whereas the occurrence of psi alone, and the combination of mu and psi, were found in only one liver each.     

Drug induction and sex differences of renal glutathione S-transferases in the rat.

Treatment of male rats with 3,4-benzopyrene, 3-methylcholanthrene and phenobarbital resulted in the induction of glutathione S-aryl- and S-aralkyl-transferase activities in kidney cytosol. Benzopyrene produced 77 and 44% increases in aryl and aralkyl activities respectively. Methylcholanthrene caused 73 and 86% increases in the retrospective activities, whereas phenobarbital treatment increased only aralkyl activity (51%). There was no effect on epoxide or alkyl glutathione S-transferase activities with these treatments. Differences were found between the specific activities of the four glutathione S-transferases in females and males, with the following female/male ratios: aryl 0.74; aralkyl 2.37; epoxide 1.52; alkyl 1.33. No changes in Km values were observed relative to drug induction or sex differences. Comparisons are made between the findings of this report and corresponding experiements with liver.     

Bile acid inhibition of basic and neutral glutathione S-transferases in rat liver.

A purification scheme is described for the neutral glutathione S-transferases of rat liver. Discontinuous sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that one of these enzymes contains a previously unidentified subunit, which has a molecular mass of 23 000 Da and has been designated Yn. Bile acids inhibited the activity of all the basic and neutral transferases investigated, but marked differences in the effects of bile acids on individual enzymes were observed. The activity of each transferase was inhibited more by lithocholate 3-sulphate than by chenodeoxycholate, which in turn was more inhibitory than cholate. The enzymes that were most sensitive to cholate inhibition were not found to be as readily inhibited as other transferases by chenodeoxycholate or lithocholate 3-sulphate. Conversely, the activity of transferase AA was more resistant to cholate, chenodeoxycholate and lithocholate 3-sulphate inhibition than was any of the other enzymes studied.     

Binding of bile acids by glutathione S-transferases from rat liver

Binding of bile acids and their sulfates and glucuronides by purified GSH S-transferases from rat liver was studied by 1-anilino-8-naphthalenesulfonate fluorescence inhibition, flow dialysis, and equilibrium dialysis. In addition, corticosterone and sulfobromophthalein (BSP) binding were studied by equilibrium and flow dialysis. Transferases YaYa and YaYc had comparable affinity for lithocholic (Kd approximately 0.2 microM), glycochenodeoxycholic (Kd approximately to 60 microM), and cholic acid (Kd approximately equal 60 microM), and BSP (Kd approximately 0.09 microM). YaYc had one and YaYa had two high affinity binding sites for these ligands. Transferases containing the Yb subunit had two binding sites for these bile acids, although binding affinity for lithocholic acid (Kd approximately 4 microM) was lower than that of transferases with Ya subunit, and binding affinities for the other bile acids were comparable to the Ya family. Sulfated bile acids were bound with higher affinity and glucuronidated bile acids with lower affinity by YaYa and YaYc than the respective parent bile acids. In the presence of GSH, binding of lithocholate by YaYc was unchanged and binding by YbYb' was inhibited. Conversely, GSH inhibited the binding of cholic acid by YaYc but had less effect on binding by YbYb'. Cholic acid did not inhibit the binding of lithocholic acid by YaYa.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.