Preparation of monoclonal antibodies to glutathione S-transferase-pi and application to immunohistochemical study

Glutathione S-transferase (GST) EC 2.5.2.18) catalyzes conjugation of reduced glutathione with hydrophobic substrates, such as S-epoxide active molecules. It participates in glutathione metabolism and the gamma-glutamyl cycle, playing an important role in detoxification and biosynthesis of many compounds. It is also known as a marker of pre-neoplasia in chemical hepatocarcinogenesis. Isoelectric focusing studies have revealed that this enzyme is composed of several isozymes, one of which, an acidic form of GST called GST-pi, has been extracted from human placenta. In this study, we prepared monoclonal antibodies (MAb) against human GST-pi from placenta. Specificity was confirmed by immunoblots of GST-pi after polyacrylamide gel electrophoresis and inhibition testing of enzyme activity by the antibody. The subclass of the antibody was IgG1 and the light chain was kappa. In light microscopic immunohistochemical studies of human placenta using the MAb, GST-pi was localized diffusely in the cytoplasm and along the apical cell membranes of syncytial cells in villi and in the cytoplasm of cytotrophoblastic cells in the basal plate. The MAb we prepared may also be useful for analyzing the enzyme's function in detoxification and biosynthesis of many compounds, as well as for oncological studies, such as diagnosis of malignant disease and localization of oncofetal proteins in malignant tissues.     

The influence of diet on the regional distribution of glutathione S-transferase activity in channel catfish intestine

There is evidence that glutathione conjugates are the major metabolites formed following systemic uptake of carcinogenic contaminants from the intestine. The effect of commercial diet versus a semi-purified diet on the distribution of glutathione S-transferase (GST) activity was examined in proximal, medial, and distal sections of catfish intestine. The bulk of GST activity with 1-chloro-2,4-dinitrobenzene, ethacrynic acid, and 3H-benzo[a]pyrene-4,5-oxide, and the percent cytosolic protein cross-reacting with anti-catfish GST-pi were in the more proximal segments and dropped off distally in the two diet groups. However, the total GST-pi cross-reacting protein in the proximal section was significantly higher in fish fed a chow diet. Western blot analysis revealed pi-class GST to be expressed principally in the proximal intestine. Cytosol samples cross-reacted with antibodies to human GST-alpha, -mu, and -pi, but not -theta, classes. Alpha-like GST isoforms of MW 26,200 and 24,600, absent in sections from fish fed a purified diet, were differentially expressed only in the distal section of chow-fed fish. These results indicate that diet significantly elicits regional differences in GST protein levels, that components of the commercial chow affect GST protein expression in the distal intestine, and that maintenance diet should be taken into consideration during dietary exposure studies.     

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.     

A distinct human testis and brain mu-class glutathione S-transferase. Molecular cloning and characterization of a form present even in individuals lacking hepatic type mu isoenzymes

mu-Class glutathione S-transferases (GSTs) were identified in all 13 human testes and 28 brains examined; even subjects whose livers were devoid of mu-GSTs expressed extrahepatic GSTs of this class. Testes and brains from individuals with mu-class GSTs in their livers had additional forms that also reflected the liver phenotypes. An isoenzyme with an isoelectric point of 5.2, which was a major GST in testis and present as well in cerebral cortex but not detected in any livers, was identified and purified. Sequence analysis of peptides derived by cleavage of the testicular mu-class GST by Achromobacter protease I revealed distinct aspects of primary structure not found previously in any mammalian mu-class GSTs. These unique features included a blocked and extended amino terminus and 3 additional residues (Pro-Val-Cys) at the carboxyl terminus. This structure was confirmed by molecular cloning and sequencing of cDNAs derived from human testis and brain libraries. In the coding region the mRNA of the brain-testis mu-class GST was 75% homologous with that of the liver form, and its 3'-untranslated sequence was mostly divergent, indicating that it is the product of a separate gene. Distinct catalytic and structural properties of the testis-brain mu-class GSTs suggest that these GSTs may be uniquely involved in blood-barrier functions common to both organs.     

Evidence for a glycoconjugate form of glutathione S-transferase pI.

The anionic form of glutathione S-transferase from human (GST pi) and rat (GST Yp) sources has been shown to exist in multiple forms which have similar molecular weights but different isoelectric points (pIs). Treatment with endoglycosidase H caused the acidic forms of GST Yp to be converted to proteins with more basic pIs as compared to the untreated control mixtures, suggesting that an N-linked mannose moiety containing acidic residues had been removed. Inability to detect these carbohydrates by techniques requiring unsubstituted vicinal hydroxyls further suggested acidic substitutions on the sugar moiety. GST pi/Yp carbohydrate modifications were also identified by differential staining procedures. These data represent the first indication that glycosylation of GST can occur. Additionally, this may offer an explanation for the often seen microheterogeneity within a class of GST isozymes.     

Purification and characterization of glutathione transferases with an activity toward nitroglycerin from human aorta and heart. Multiplicity of the human class Mu forms

Although recent studies suggest involvement of glutathione transferase (GST) of blood vessels in vasodilation by nitroglycerin, GST forms in blood vessels remain to be studied. In this study, three GST forms (pI values 8.3, 6.6, and 4.8) were purified from human aorta and four (pI values 6.0, 5.6, 5.3, and 4.6) from the heart by affinity chromatography followed by chromatofocusing. The major form of both aorta (pI 4.8) and heart (pI 4.6) was identified as GST-pi, and the other five forms were immunologically related to GST-mu, suggesting that the five belong to the Mu class. Among nine human GST forms, including three in the Alpha class purified from the liver, GST-mu, aorta pI 8.3 form, and GST-I (a form of the Alpha class, corresponding to GST-epsilon (B1B1)) showed high activities toward nitroglycerin, 1.08, 0.85, and 0.78 units/mg protein, respectively. GST-pi did not exhibit the activity. The Km values of the aorta form (pI 8.3) for glutathione (GSH) and nitroglycerin were calculated as 0.12 and 1.1 mM, respectively. The Km values of GST-mu and GST-I for GSH were 0.29 and 0.09 mM, and those for nitroglycerin were 2.5 and 0.3 mM, respectively. The activity of the pI 8.3 form as well as GST-mu toward nitroglycerin was inhibited by bromosulfophthalein, which is known to inhibit the relaxation of rabbit aorta induced by nitroglycerin, at the lower concentration (IC50, 2 microM) than was GST-I (IC50, 32 microM). Two-dimensional gel electrophoresis and N-terminal amino acid sequence analysis revealed that five forms in the Mu class are homo- or heterodimers of five different subunits named M1 (pI 7.0/Mr 27,000), M2 (6.6/27,000), M3 (6.0/27,000), N1 (6.5/26,500), and N2 (5.9/26,500). The subunit structures of the five forms are as follows: pI 8.3 form, M1M2; 6.6 form, M2N1; 6.0 form, M3M3; 5.6 form, M3N2; and 5.3 form, N2N2. M3 and N2 seem to correspond to the subunits of GST-mu, and -4 (Board, P. G., Suzuki, T., and Shaw, D. C. (1988) Biochim. Biophys. Acta 953, 214-217), respectively. These subunits except N1 are different from each other at two or three positions in the first 20 residues of N-terminal amino acid sequence. These results indicate the presence of five different subunits in the human Mu class and also suggest that GST-M1M2 and -M2N1 found in the aorta are involved in the expression of the pharmacologic effect of nitroglycerin.     

The tyrosine kinase activity of the epidermal-growth-factor receptor is necessary for phospholipase A2 activation.

Cytosolic glutathione transferases (GSTs) were purified from the rat spleen by S-hexyl-GSH-Sepharose chromatography, and two major forms were identified as GSTs 2-2 and 7-7 (GST P). Besides these forms an acidic form (pI 5.8) was purified by chromatofocusing at pH 7-4 and it accounted for about 1% of the total GST activity bound to S-hexyl-GSH-Sepharose. Two-dimensional gel electrophoresis revealed that it is a homodimer (subunit Mr 26,000 with pI 5.8). Immunoblot analysis demonstrated that it was immunologically related to GSTs 2-2 and 1-1, and its N-terminal amino acid was apparently blocked, similarly to other forms of the class Alpha. This form had a low activity towards cumene hydroperoxide or 4-hydroxynon-2-enal, indicating that this form differed from GSTs 10-10 and 8-8 as well as from GSTs 1-1 and 2-2. These results suggest that it is a new form of GST belonging to the class Alpha.     

Purification of a new acidic glutathione S-transferase, GST-Yn1Yn1, with a high leukotriene-C4 synthase activity from rat brain

A new acidic form of glutathione S-transferase (GST, pI 6.2) was purified from rat brain by S-hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20-25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (MT or 6-6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the MT in isoelectric point, activity towards 1,2-dichloro-4-nitrobenzene and immunological properties. On two-dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST-MT. Therefore, the brain acidic form is a homodimer, and named GST-Yn1Yn1. The activity was inhibited by sulfasalazine, an inhibitor of leukotriene-C4 synthase. This form (GST-Yn1Yn1) showed the highest leukotriene-C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 microM and 3.5 mM respectively. A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST-P (7-7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST-P also showed the significant leukotriene-C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotriene A4 was 100 microM, fourfold higher than that of GST-Yn1 Yn1. These results suggest that mainly GST-Yn1 Yn1 may be involved in leukotriene-C4 synthesis in rat brain.     

Amino acid sequence of the major form of toad liver glutathione transferase

To investigate structural relationship between amphibian and mammalian GSTs the complete amino acid sequence of the major form of glutathione transferase present in toad liver (Bufo bufo) was determined. The enzyme subunit is composed of 210 amino acid residues corresponding to a molecular mass of 24,178 Da. In comparison with the primary structure of amphibian bbGSTP1-1, toad liver GST showed 54% sequence identity. On the other hand, toad liver GST showed about 45-55% sequence identity when compared with other pi class GST and less then 25% identity with GST of other classes. Amino acid residues involved in the H site and in the key and lock structure of the toad enzyme are significantly different from those of bbGSTP1-1 and other mammalian pi class GST. On the basis of its structural and immunological properties the toad liver GST, indicated as bbGSTP2-2, could represent the prototype of a subset of the pi family.     

Interaction of 1,4-benzoquinone and 2,4-dichlorophenoxyacetic acid with microsomal glutathione transferase from rat liver

Glutathione transferase (GST) was purified from the microsomes of rat liver by glutathione affinity chromatography. The interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1,4-benzoquinone with microsomal GST was investigated and compared with cytosolic GST. The kinetic inhibition pattern of 1,4-benzoquinone towards microsomal GST was found to be different from that towards cytosolic GST. Microsomal GST purified by affinity chromatography was inhibited by 2,4-D in a non dose-dependent manner, while the crude microsomal GST was inhibited in a dose-dependent manner. This difference was shown to be induced by a reaction on the affinity column, and not by Triton X-100 (also shown to be a GST inhibitor), glutathione, or the elution buffer 0.2% Triton X-100 and 5 mM glutathione in 50 mM Tris-HCl, pH 9.6. The binding of microsomal GST to the affinity matrix caused a partial inactivation of the active site for 2,4-D interaction. The results show that the properties of soluble GST enzymes may not be extrapolated to the microsomal ones.     

The isolation, characterisation and kinetics of glutathione-S-transferase from human platelets

Glutathione S-transferase activity from human platelets was purified to homogeneity by affinity chromatography. The purified enzyme was found to be the acidic form and its molecular and catalytic properties were identical to acidic glutathione S-transferases purified from other human tissues. The purified platelet enzyme had no peroxidase activity and did not protect microsomes against peroxidation.     

Soluble glutathione transferase isoenzymes in Daphnia magna straus and their interaction with 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone

The glutathione transferase (GST) activity in the cytosol of the water flea Daphnia magna Straus was partially purified by glutathione affinity chromatography. Chromatofocusing on the Polybuffer exchangers 94 and 118 separated the GST isoenzymes in one neutral and four cationic forms, and some minor fractions one of which was an anionic form. The major GST isoenzymes were partially characterized by different biochemical parameters. The water pollutants 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone inhibited the water flea GST isoenzymes, following the same kinetic inhibition patterns as for rat liver GST. It is concluded that water flea GST can play an important role in the detoxification of aquatic pollutants.     

The use of acetylcholinesterase activity in Ruditapes decussatus and Mytilus galloprovincialis in the biomonitoring of Bizerta lagoon

Glutathione S-transferases (GST) form an important family of biotransformation enzymes catalyzing the conjugation of glutathione to a great variety of xenobiotic compounds. The objective of this study was to compare the different characteristics of GST from freshly isolated rainbow trout hepatocytes with those corresponding to the total liver of the same fish, in order to establish the similarities. GST was purified by affinity chromatography and enzymatic activity was determined towards two substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETHA). The different isoenzymes were determined by HPLC associated with SDS-PAGE. Slight differences between the samples were obtained when the results corresponding to the enzyme activity were compared. HPLC results showed that all GST isoforms present in the total liver samples were represented in the isolated cells too, corresponding to isoforms with molecular masses of approximately 25.5 and 23.0 kDa.     

Characterization and molecular cloning of a glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae).

A glutathione S-transferase (GST) was purified from the larval cattle tick, Boophilus microplus (Acari: Ixodidae), by glutathione-affinity chromatography. The purified enzyme appeared as a single band on SDS-PAGE and has a molecular mass of 25.8 kDa determined by mass spectrometry. The N-terminus of the purified enzyme was sequenced. The full-length cDNA of the enzyme was isolated by RT-PCR using degenerate oligonucleotides derived from the N-terminal amino acid sequence. The cDNA contains an open reading frame encoding a 223-amino-acid protein with the N-terminus identical to the purified GST. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian mu class GST.     

The major glutathione S-transferase in salmonid fish livers is homologous to the mammalian pi-class GST.

1. The hepatic glutathione S-transferase (GST) isoenzymes were isolated and characterized from salmon, sea trout and rainbow trout. 2. In all three species the predominant GST expressed comprised subunits of Mr 24,800. These subunits each co-migrated with the rat pi-class Yf polypeptide during SDS/polyacrylamide gel electrophoresis. 3. Western blotting experiments demonstrated immunochemical cross-reactivity between the major salmonid and the rat pi-class GSTs. 4. The salmon GST of subunit Mr 24,800 was digested with cyanogen bromide and the peptides, once purified by reverse-phase HPLC, were subjected to automated amino acid sequencing. 5. Over the region sequenced, the salmon GST possessed about 65% homology with the rat and human pi-class GST.     

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.     

Human microsomal glutathione S-transferase. Its involvement in the conjugation of hexachlorobuta-1,3-diene with glutathione.

A microsomal glutathione S-transferase (GST) was purified from human liver. This enzyme was shown to have characteristics similar to those of the rat microsomal GST described by Morgenstern & De Pierre [(1983) Eur. J. Biochem. 134, 591-597]. The specific activity of human microsomal GST towards 1-chloro-2,4-dinitrobenzene or cumene hydroperoxide can be stimulated by treating the enzyme with N-ethylmaleimide. This enhancement of activity is accompanied by increased sensitivity to inhibition by haematin and cholic acid. The subunit Mr values of the rat and human enzymes are similar (approx. 17,300), and the proteins are immunologically related. During purification, both human and rat microsomal GST enzymes are the only hepatic proteins obtained from Triton X-100-solubilized microsomal fractions that show activity towards the nephrotoxin hexachlorobuta-1,3-diene. The involvement of microsomal GST in toxification reactions is discussed.     

Molecular cloning, sequencing, and expression of human myocardial fatty acid ethyl ester synthase-III cDNA

Fatty acid ethyl ester synthase-III (FAEES-III), previously purified to homogeneity from human heart, metabolizes ethanol nonoxidatively. Using a derived partial amino acid sequence and corresponding oligonucleotide probes, the cDNA for this enzyme has been cloned from a human heart lambda gtll library. Of the five positive clones obtained, one contained a complete coding region (630 base pairs) and the entire 3'-noncoding region (41 base pairs). From this nucleotide sequence the complete 210 amino acid sequence of FAEES-III (Mr 23,307) is reported. Comparison of its amino acid sequence with that of glutathione S-transferase pi-1 suggests that they belong to the same gene family since they differ in only six nucleotides and four amino acids. The sequence of FAEES-III was also compared with those of placental glutathione S-transferase and the basic glutathione S-transferase. FAEES-III was 84% homologous with placental glutathione S-transferase but only less than 10% homologous with the basic glutathione S-transferase. Northern blots demonstrate expression of FAEES-III mRNA in normal human liver, placenta, and heart. In all cases, the mRNA for the enzyme is 0.7 kilobase in size. MCF-7 cells transfected with FAEES-III cDNA have a 14-fold increase in synthase activity and a 12-fold increase in glutathione S-transferase (GST) activity compared with control cells. MCF-7 cells transfected with GST pi-1 cDNA have a 13-fold increase in GST activity compared with control cells but no increase in synthase activity. When the supernatant of COS-7 cells transfected with FAEES-III cDNA were immunoblotted with rabbit FAEES-III antibody, a band at 24 kilodaltons was demonstrated. Thus, we have obtained the first cDNA and amino acid sequence for a human FAEES-III which also has significant GST activity, and we have identified 4 residues potentially responsible for conferring ethanol recognition to GSTs.     

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

Molecular characterization of corn glutathione S-transferase isozymes involved in herbicide detoxication

Corn ( Zea mays L.) glutathione S-transferases (EC 2.5.1.18) have attracted interest, in part, due to their involvement in the metabolism of several herbicides, including atrazine and alachlor. Three corn, glutathione S-transferases have been purified, and cDNA clones have been isolated and sequenced for two of these, GST I and GST III. In addition to showing some amino acid sequence similarity to each other, the two sequenced corn glutathione S-transferases also show some similarity to rat and human enzymes. The corn glutathione S-transferases responsible for atrazine tolerance have not yet been purified or cloned, but purification attempts indicate that corn has two glutathione S-transferases with activity towards atrazine. While many glutathione S-transferases from various organisms have been detected by using 1-chloro-2,4-dinitrobenzene as a substrate, the atrazine-specific glutathione S-transferases have very little or no activity with 1-chloro-2,4-dinitrobenzene. This shows the importance of assaying with a variety of substrates when characterizing glutathione S-transferases.