Purification and characterization of human muscle glutathione S-transferases: evidence that glutathione S-transferase zeta corresponds to a locus distinct from GST1, GST2, and GST3.

Human muscle glutathione S-transferase isozyme, GST zeta (pI 5.2) has been purified by three different methods using immunoaffinity chromatography, DEAE cellulose chromatography, and isoelectric focusing. GST zeta prepared by any of the three methods does not recognize antibodies raised against the alpha, mu, or pi class glutathione S-transferases of human tissues. GST zeta has a blocked N-terminus and its peptide fingerprints also indicate it to be distinct from the alpha, mu, or pi class isozymes. As compared to GSTs of alpha, mu, and pi classes, GST zeta displays higher activities toward t-stilbene oxide and Leukotriene A4 methyl ester. GST zeta also expresses GSH-peroxidase activity toward hydrogen peroxide. The Kms of GST zeta for CDNB and GSH were comparable to those reported for other human GSTs but its Vmax for CDNB, 7620 mol/mol/min, was found to be considerably higher than that reported for other human GSTs. The kinetics of inhibition of GST zeta by hematin, bile acids, and other inhibitors also indicate that it was distinct from the three classes of GST isozymes. These studies suggest that GST zeta corresponds to a locus distinct from GST1, GST2, and GST3 and probably corresponds to the GST4 locus as suggested previously by Laisney et al. (1984, Human Genet. 68, 221-227). The results of peptide fingerprints and kinetic analysis indicate that as compared to the pi and alpha class isozymes, GST zeta has more structural and functional similarities with the mu class isozymes. Besides GST zeta several other GST isozymes belonging to pi and mu class have also been characterized in muscle. The pi class GST isozymes of muscle have considerable charge heterogeneity among them despite identical N-terminal sequences.     

Glutathione S-transferases of human lung: characterization and evaluation of the protective role of the alpha-class isozymes against lipid peroxidation.

Glutathione S-transferase (GST) isozymes of human lung have been purified, characterized, quantitated, and, based on their structural and immunological profiles, identified with their respective classes. The tau-, mu-, and alpha-class GSTs represented 94, 3, and 3% activities of total human lung GSTs toward CDNB, respectively, and 60, 10, and 30% of total GST protein, respectively. Both the mu- and the alpha-class GSTs of human lung exhibited heterogeneity. The two mu-class GSTs of human lung had pI values of 6.5 and 6.25 and were differentially expressed in humans. Significant differences were seen between the kinetic properties of these two isozymes and also between the lung and liver mu-class GSTs. The alpha-class GST isozymes of lung resolved into three peaks during isoelectric focusing corresponding to pI values of 9.2, 8.95, and 8.8. All three alpha-class GSTs isozymes had blocked N-termini and were immunologically similar to human liver alpha-class GSTs. Peptide fingerprints generated by SV-8 protease digestion and CNBr cleavage indicated minor structural differences between the liver and the lung alpha-class GSTs. The three alpha-class GSTs of lung expressed glutathione peroxidase activities toward the hydroperoxides of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, with Km values in the range of 22 to 87 microM and Vmax values in the range of 67-120 mol/mol/min, indicating the involvement of the alpha-class GSTs in the protection mechanisms against peroxidation. All three classes of lung GSTs expressed activities toward leukotriene A4 methyl ester and epoxy stearic acid but the mu-class GSTs had relatively higher activities toward these substrates.     

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.     

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.     

Ligand-based protein alignment and isozyme specificity of glutathione S-transferase inhibitors

Glutathione S-transferases (GST, E.C.2.5.1.18) comprise a family of detoxification enzymes. Elevated levels of specific GST isozymes in tumor cells are thought responsible for resistance to chemotherapeutics, which renders selective GST inhibitors potentially useful pharmaceutical agents. We discuss the development of a structure activity model that rationalizes the isozyme selectivity observed in a series of 12 glutathione (GSH) analogues. Enzymatic activity data was determined for human P1-1, A1-1, and M2-2 isozymes, and these data were then considered in light of structural features of these three GST proteins. A survey of all GST structures in the PDB revealed that GSH binds to these proteins in a single “bioactive” conformation. To focus on differences between binding sites, we exploited our finding of a common GSH conformation and aligned the GST x-ray structures using bound ligands rather than the backbones of the different proteins. Once aligned, binding site lipophilicity and electrostatic potentials were computed, visualized, and compared. Docking and energy minimization exercises provided additional refinements to a model of selectivity developed initially by visual analysis. Our results suggest that binding site shape and lipophilic character are key determinants of GST isozyme selectivity for close GSH analogues. Proteins 28:202–216, 1997. © 1997 Wiley-Liss Inc.     

Partial Characterization of Glutathione S-Transferase Isozymes Induced by the Herbicide Safener Benoxacor in Maize

The effects of the dichloroacetamide safener benoxacor on maize (Zea mays L. var Pioneer 3906) growth and glutathione S-transferase (GST) activity were evaluated, and GST isozymes induced by benoxacor were partially separated, characterized, and identified. Protection from metolachlor injury was closely correlated with GST activity, which was assayed with metolachlor as a substrate, as benoxacor concentration increased from 0.01 to 1 [mu]M. GST activity continued to increase at higher benoxacor concentrations (10 and 100 [mu]M), but no further protection was observed. Total GST activity with metolachlor as a substrate increased 2.6- to 3.8-fold in response to 1 [mu]M benoxacor treatment. Total GST activity from maize treated with or without 1 [mu]M benoxacor was resolved by fast protein liquid chromatography anion-exchange chromatography into four major activities, designated activity peaks A, B, C, and D in their order of elution. These GST activity peaks were enhanced to varying degrees by benoxacor. Activity peak B showed the least induction, whereas activity peak A was absent constitutively and thus highly induced by benoxacor. In contrast to earlier reports, there appear to be not one, but at least two, major constitutive isozymes (activity peaks A and D) having activity with metolachlor as substrate; there were at least three such isozymes in benoxacor-treated maize (activity peaks A, C, and D). The elution volumes of activity peaks A, B, C, and D were compared with those of partially purified maize GST I and GST II; also, the reactivity of polypeptides in these activity peaks with antisera to GST I or GST I/III (mixture) was evaluated. Evidence from these experiments indicated that activity peak B contained GST I, and activity peak C contained GST II and GST III. Activity peaks A and D contained unique GSTs that may play a major role in metolachlor metabolism and in the safening activity of benoxacor in maize. Isozymes present in activity peaks A and D were not detected in earlier reports because of the very low activity with the artificial substrate 1-chloro-2,4-dinitrobenzene. Immunoblotting experiments also indicated the presence of numerous unidentified GST subunits, including multiple subunits in chromatography fractions containing single peaks of GST activity; this is indicative of the likely complexity and diversity of the maize GST enzyme family.     

Glutathione S-transferase isozymes in Aedes aegypti: Purification,characterization, and isozyme-specific regulation

Glutathione S-transferase (GST) isozymes were purified from the GG strain of Aedes aegypti, a strain having ≥4-fold higher total GST activity compared to the wild-type lab strain. Purification involved S-hexyl-glutathione affinity chromatography in high salt buffer, and GST specific elution with S-(p-bromobenzyl)-glutathione. Final purification was accomplished on DEAE-Sepharose. Two isozymes, GST-1b and GST-2 were purified using this procedure, and an additional isozyme, GST-1a, was partially purified. The GST-2 isozyme has one of the highest specific activities reported for a GST, with a specific activity of 739 μmol/min/mg using 1-chloro-2,4-dinitrobenzene (CDNB), and 16.4 μmol/min/mg using 3,4-dichloronitrobenzene (DCNB) as substrates. GST-2, GST-1a, and GST-1b were analyzed for amino acid composition and subjected to N-terminal sequencing. All three GSTs showed amino acid differences, especially among the nonpolar and polar amino acids. The amino acid composition of GST-1b was found to be more similar to GST 1-1 from Drosophila melanogaster than to GST-2 or GST-1a from Aedes aegypti. Only GST-2 gave N-terminal sequence data, raising the possibility that GST-1a and 1b are N-terminally blocked. The A. aegypti GST-2 showed amino acid sequence identity or similarity in all but one residue between residue numbers 31 through 41 compared to the D. melanogaster and Musca domestica GST 1-1 isozymes. The pattern of GST isozyme expression was analyzed in various tissues and stages of development of the GG and wild type strains using isozyme-specific antisera and substrates. GST-1a was constitutively overexpressed in all tissues examined in the GG strain compared to the wild type strain. The expression of GST-1b was similar in both strains for all tissues and developmental stages examined. GST-2 was constitutively overexpressed in head, thorax and abdomen, but was not detected in ovaries of the GG strain. These results suggest that elevated GST activity in the GG strain is due to constitutive overexpression of GST-2 and GST-1a. GST-1a, GST-1b and GST-2 apparently are the products of 3 independently regulated genes and appear to be expressed in a tissue-specific manner.     

Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis

Four isozymes of pyruvate kinase are differentially expressed in human tissue. Human pyruvate kinase isozyme M2 (hPKM2) is expressed in early fetal tissues and is progressively replaced by the other three isozymes, M1, R, and L, immediately after birth. In most cancer cells, hPKM2 is once again expressed to promote tumor cell proliferation. Because of its almost ubiquitous presence in cancer cells, hPKM2 has been designated as tumor specific PK-M2, and its presence in human plasma is currently being used as a molecular marker for the diagnosis of various cancers. The X-ray structure of human hPKM2 complexed with Mg(2+), K(+), the inhibitor oxalate, and the allosteric activator fructose 1,6-bisphosphate (FBP) has been determined to a resolution of 2.82 A. The active site of hPKM2 is in a partially closed conformation most likely resulting from a ligand-induced domain closure promoted by the binding of FBP. In all four subunits of the enzyme tetramer, a conserved water molecule is observed on the 2-si face of the prospective enolate and supports the hypothesis that a proton-relay system is acting as the proton donor of the reaction (1). Significant structural differences among the human M2, rabbit muscle M1, and the human R isozymes are observed, especially in the orientation of the FBP-activating loop, which is in a closed conformation when FBP is bound. The structural differences observed between the PK isozymes could potentially be exploited as unique structural templates for the design of allosteric drugs against the disease states associated with the various PK isozymes, especially cancer and nonspherocytic hemolytic anemia.     

Purification and characterization of glutathione reductase isozymes specific for the state of cold hardiness of red spruce.

Isozymes of glutathione reductase (GR) have been purified from red spruce (Picea rubens Sarg.) needles. Two isozymes could be separated by anion-exchange chromatography from both nonhardened or cold-hardened tissue. Based on chromatographic elution profiles, the isozymes were designated GR-1NH and GR-2NH in preparations from nonhardened needles, and GR-1H and GR-2H in preparations from hardened needles. N-terminal sequencing and immunological data with antisera obtained against GR-1H and GR-2H established that the isozymes from hardened needles are different gene products and show significant structural differences from each other. Chromatographic, electrophoretic, and immunological data revealed only minor differences between GR-2NH and GR-2H, and it is concluded that these isozymes are very similar or identical. Anion-exchange chromatography and native polyacrylamide gel electrophoresis also established that GR-1NH and GR-1H are different proteins. From these data we conclude that GR-1H is a distinct gene product, present only in hardened needles. Therefore, GR-1H can be considered to be a cold-hardiness-specific GR isozyme, and GR-1NH can be considered to be specific for nonhardened needles. It is proposed that GR-1H is a cold-acclimation protein.     

Purification and biochemical characterization of the rabbit pulmonary glutathione S-transferase: stereoselectivity and activity toward pyrene 4,5-oxide

Two homodimeric isozymes, glutathione S-transferase (GST) 25 kDa and GST 27 kDa, in equal proportion comprise the majority (greater than 75%) of the pulmonary cytosolic GST of untreated rabbits. The subunits of GST 25 kDa and GST 27 kDa are distinguishable by electrophoretic mobility (25 and 27 kDa, respectively), apparent isoelectric points (pI 7.4 and pI 9.1, respectively), and immunoreactivity. Immunoblots indicated that these subunits may be minor components in hepatic cytosol. The pulmonary isozymes could not be distinguished by their activities toward chloro-2,4-dinitrobenzene (CDNB) or activity and stereoselectivity toward pyrene 4,5-oxide (PyO). The purified GST fractions represented less than or equal to 16% of the PyO activity for pulmonary cytosol. The stereoselectivity of the cytosolic GST for the pro-S-configured oxirane carbon of PyO was not maintained in the purified preparations which were virtually nonstereoselective. Immunoprecipitation of pulmonary cytosolic GST with anti-GST 27 kDa and anti-GST 25 kDa indicated that at least 84 and 60% of the activity toward CDNB and PyO, respectively, is mediated by the two isozymes. The specific PyO activities of GST 27 kDa, GST 25 kDa, and the rabbit hepatic preparations (approximately 0.2 unit/mg) were similar to that of hepatic GST purified from horse, cow, and pig, and to human placental GST pi (0.02-0.5 unit/mg) but one-tenth that of rat hepatic GST or human hepatic GST mu. However, the activity of the hepatic cytosol from rat and human was similar to that of rabbit. Thus, some GST isozymes may be particularly susceptible to modulation of activity/stereoselectivity that can be discerned with arene oxide substrates such as PyO.     

Protective effect of aqueous extract of Phyllanthus fraternus against bromobenzene induced changes on cytosolic glutathione S-transferase isozymes in rat liver

The aim of this study was to investigate beneficial effect of aqueous extract of Phyllanthus fraternus (AEPF) on bromobenzene (BB) induced changes on cytosolic glutathione S-transferase (GST) isozymes in rat liver. Administration of BB significantly decreased the activity of GST, however, prior administration of AEPF prevented the BB induced decrease in GST activity. Further the cytosolic GSTs were purified from 3 groups of animals (control, BB and AEPF+BB administered) and resolved into three protein bands on SDS-PAGE. Densitometric analysis showed a significant decrease in BB group compared to control. Further, 2D PAGE analysis resolved these proteins into 8 bands which were identified as five isozymes of alpha, two of Mu and one of theta by MALDI-TOF MS and also observed decreased levels of isozymes in BB group. However, on prior administration of AEPF significantly prevented the BB induced decrease in GSTs and restored to normal levels.     

Nonspecific esterase isozymes that focus at identical isoelectric points are kinetically analogous

Isoelectric focusing (IEF) of soluble nonspecific esterases of rat kidney and testis exhibits an identical array of organophosphate-resistant cathodal isozymes. To ascertain whether such isozymes that focus at the same pI are also kinetically analogous, two isozymes, both focused at pI 7.2, were isolated, one from each organ, by elution from cut-out, unstained gel segments. Although the esterases of the whole soluble fraction of kidney and testis exhibited different kinetic properties and organophosphate susceptibility, no differences were observed with regard to the isozymes. Therefore, because of similar electrophoretic and kinetic behavior, the two isozymes can be regarded as phenotypic expressions of similar genetic products.     

Structure of human brain fructose 1,6-(bis)phosphate aldolase: linking isozyme structure with function

Fructose-1,6-(bis)phosphate aldolase is a ubiquitous enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-(bis)phosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceral-dehyde-3-phosphate or glyceraldehyde, respectively. Vertebrate aldolases exist as three isozymes with different tissue distributions and kinetics: aldolase A (muscle and red blood cell), aldolase B (liver, kidney, and small intestine), and aldolase C (brain and neuronal tissue). The structures of human aldolases A and B are known and herein we report the first structure of the human aldolase C, solved by X-ray crystallography at 3.0 A resolution. Structural differences between the isozymes were expected to account for isozyme-specific activity. However, the structures of isozymes A, B, and C are the same in their overall fold and active site structure. The subtle changes observed in active site residues Arg42, Lys146, and Arg303 are insufficient to completely account for the tissue-specific isozymic differences. Consequently, the structural analysis has been extended to the isozyme-specific residues (ISRs), those residues conserved among paralogs. A complete analysis of the ISRs in the context of this structure demonstrates that in several cases an amino acid residue that is conserved among aldolase C orthologs prevents an interaction that occurs in paralogs. In addition, the structure confirms the clustering of ISRs into discrete patches on the surface and reveals the existence in aldolase C of a patch of electronegative residues localized near the C terminus. Together, these structural changes highlight the differences required for the tissue and kinetic specificity among aldolase isozymes.     

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.     

Glutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor gamma (PPAR gamma) ligand, 15-deoxy-delta 12,14prostaglandin J2 (15-d-PGJ2)

15-Deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), a terminal metabolite of the J-series cyclopentenone prostaglandins, influences a variety of cellular processes including gene expression, differentiation, growth, and apoptosis. As a ligand of peroxisomal proliferator-activated receptor gamma (PPAR gamma), 15-d-PGJ(2) can transactivate PPAR gamma-responsive promoters. Previously, we showed that multidrug resistance proteins MRP1 and MRP3 attenuate cytotoxic and transactivating activities of 15-d-PGJ(2) in MCF7 breast cancer cells. Attenuation was glutathione-dependent and was associated with formation of the glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, and its active efflux by MRP. Here we have investigated whether the glutathione S-transferases (GST) can influence biological activities of 15-d-PGJ(2). MCF7 cells were stably transduced with human cytosolic GST isozymes M1a, A1, or P1a. These GSTs had no effect on 15-d-PGJ(2) cytotoxicity when expressed either alone or in combination with MRP1. However, expression of any of the three GSTs significantly inhibited 15-d-PGJ(2)-dependent transactivation of a PPAR gamma-responsive reporter gene. The degree of inhibition correlated with the level of GST expressed. Under physiologic conditions, the nonenzymatic rate of 15-d-PGJ(2) conjugation with glutathione was significant. Of the three GST isozymes, only GSTM1a-1a further stimulated the rate of 15-d-PGJ(2)-SG formation. Moreover, GSTM1a-1a rate enhancement was only a transient burst that was complete within 15 s. Hence, catalysis plays little, if any, role in GST inhibition of 15-d-PGJ(2)-dependent transactivation. In contrast, inhibition of transactivation was associated with strong GST/15-d-PGJ(2) interactions. Potent inhibition by 15-d-PGJ(2) and 15-d-PGJ(2)-SG of GST activity was observed with K(i) in the 0.15-2.0 microM range for the three GST isozymes, results suggesting avid associations between GST and 15-d-PGJ(2) or 15-d-PGJ(2)-SG. Electrospray ionization mass spectrometry (ESI/MS) studies revealed no stable adducts of GST and 15-d-PGJ(2) indicating that GST/15-d-PGJ(2) interactions are primarily noncovalent. These results are consistent with a mechanism of GST-mediated inhibition of transactivation in which GST binds 15-d-PGJ(2) and 15-d-PGJ(2)-SG thereby sequestering the ligands in the cytosol away from their nuclear target, PPAR gamma.     

Biochemical differences between alcohol dehydrogenases of Drosophila melanogaster.

This paper describes substrate specificities, developmental changes in activity, pH profiles, and heat stabilities of isozymes produced by four Adh genotypes in D. melanogaster. No differences are found in the substrate specificities of isozymes from the different genotypes but studies of the other three properties reveal significant differences between the isozymes. Thus relatively low activities are found among extracts of AdhF Adhn2 larvae and among extracts of AdhF AdhF adults aged 44 days. Also AdhF AdhS and AdhS AdhS extracts have relatively high activities at pH 6-5, and AdhF Adhn2 extracts have relatively low activities at pH values above 10-0. Finally, extracts of AdhF AdhF and AdhF AdhS are more stable at 40 degrees C than are those of AdhS AdhS and AdhF Adhn2.     

Isolation and comparative analysis of multiple isoenzymes of glutathione-S-transferase from the liver of intact rats and rats administered phenobarbital, 3-methylcholanthrene and butylhydroxytoluene

Seven glutathione-S-transferase (GST) isozymes were purified from liver cytosol of intact male Wistar rats: 1-1(A), 1-1(B), 1-2, 2-2, 3-3, 3-4, 4-4. Treatment of rats with butylated hydroxytoluene (BHT) led to the induction of isozymes GST 1-1(A), 1-1(B) (2-fold), 3-3 (3.5-fold) as well as to the appearance of two new isozymes--1-3 and 4-4(A). Phenobarbital (PB) induced isozymes GST 1-1(A), 1-1(B) (2-fold) and 3-3 (1.5-fold). BHT and PB caused an increase in the specific activity of isozymes 1-1(A), 1-1(B), 3-3, 3-4 towards 1-chloro-2.4-dinitrobenzene and 1.2-dichloro-4-nitrobenzene. 3-Methylcholanthrene (MC) induced isozymes 1-2 (1.5-fold), 2-2 (2-fold) and 4-4 (3-fold). A conclusion was drawn that BHT and PB induced the GST subunits 1 and 3, whereas MC--subunits 2 and 4.     

Sex difference in subunit composition of hepatic glutathione S-transferase in rats

The activities of hepatic cytosolic glutathione S-transferases (GSTs) towards 1,2-dichloro-4-nitrobenzene in male rats were higher than those in females, however, the enzyme activities towards 1-chloro-2,4-dinitrobenzene were not significantly different between the two sexes. SDS-PAGE analysis of GSTs purified from male and female rat hepatic cytosols by affinity column chromatography showed that there was a significant difference in the subunit composition between the two sexes. With regard to the several isozymes of GSTs in male and female rats, isozymes with basic and neutral/acidic isoelectric points were separated into seven molecular species by chromatofocusing. These sex differences in the quantitative proportions of GST isozymes were also confirmed by immunotitration using anti-GST-BL and -AC antibodies. On the other hand, glutathione peroxidase (GSH-Px) activities in rat hepatic cytosol towards hydrogen peroxide and cumene hydroperoxide were markedly higher in females than in males. Of the two types of GSH-Px, selenoenzyme (Se-GSH-Px) and the Se-independent enzyme (non-Se-GSH-Px), the former was found to be mainly responsible for the sex difference in the enzyme activities. Moreover, the GSH-Px activity of GSTs, non-Se-GSH-Px, was also higher in females than that in males. Since GST isozymes of the BL type are known to possess GSH-Px activity towards cumene hydroperoxide, the increased activities of non-Se-GSH-Px in the female hepatic cytosol seemed to be mainly due to the increased transferase activities of the isozymes, GST-L2 and -BL.