Conjugation of the linoleic acid oxidation product, 13-oxooctadeca-9,11-dienoic acid,a bioactive endogenous substrate for mammalian glutathione transferase

The oxidation of linoleic acid leads to the generation of several products with biological activity, including 13-oxooctadeca-9,11-dienoic acid (13-OXO), a bioactive 2,4-dienone that has been linked to cell differentiation. In the current work, the conjugation of 13-OXO by human glutathione transferases (GSTs) of the alpha (A1–1, A4–4), mu (M1–1, M2–2) and pi (the allelic variants P1–1/ile, and P1–1/val) classes, and a rat theta (rT2–2) class enzyme has been evaluated. The kinetics and stereoselectivity of the production of the 13-OXO-glutathione conjugate (13-OXO-SG) have been examined. In contrast to many xenobiotic substrates, the endogenous substrate 13-OXO does not exhibit an appreciable non-enzymatic rate of conjugation under physiological conditions. Therefore, the GST-catalyzed conjugation takes on greater significance as it provides the only realistic means for formation of 13-OXO-SG in most biological systems. Alpha class enzymes are most efficient at catalyzing the formation of 13-OXO-SG with k_cat/K_m values of 8.9 mM⁻¹ s⁻¹ for GST A1–1 and 2.14 mM⁻¹ s⁻¹ for GST A4–4. In comparison, enzymes from the mu and pi classes exhibit specificity constants from 0.4 to 0.8 mM⁻¹ s⁻¹. Conjugation of 13-OXO with glutathione at C-9 of the substrate can yield a pair of diastereomers that can be resolved by chiral HPLC. GSTs from the mu and pi classes are the most stereoselective enzymes and there is no apparent relationship between catalytic efficiency and stereoselectivity. The role of GST in the metabolic disposition of the bioactive oxidation products of linoleic acid has implications for the regulation of normal cellular functions by these versatile enzymes.     

Metabolism of oxidized linoleic acid by glutathione transferases: peroxidase activity toward 13-hydroperoxyoctadecadienoic acid

The oxidation of linoleic acid produces several products with biological activity including the hydroperoxy fatty acid 13-hydroperoxyoctadecadienoic acid (13-HPODE), the hydroxy fatty acid 13-hydroxyoctadecadienoic acid (13-HODE), and the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). In the present work, the peroxidase activity of glutathione transferases (GST) A1-1, M1-1, M2-2, and P1-1(Val 105) toward 13-HPODE has been examined. The alpha class enzyme is the most efficient peroxidase while the two enzymes from the mu class exhibit weak peroxidase activity toward 13-HPODE. It was also determined that the conjugated diene 13-HODE is not a substrate for GST from the alpha and mu classes but that 13-HODE does inhibit the GST-catalyzed conjugation of CDNB by enzymes from the alpha, mu, and pi classes. Finally, both 13-HODE and 13-OXO were shown to be inducers of GST activity in HT-29 and HCT-116 colon tumor cells. These data help to clarify the role of GST in the metabolic disposition of linoleic acid oxidation products.     

Glutathione S-transferase-catalyzed conjugation of 9,10-epoxystearic acid with glutathione

The possible role of glutathione S-transferases (GST) in detoxification of fatty acid epoxides generated during lipid peroxidation has been evaluated. Present studies showed that cytosolic human glutathione S-transferases belonging to alpha, mu, and pi classes isolated from human liver and lung catalyzed the conjugation of glutathione and 9,10-epoxystearic acid. The product of enzymatic reaction, i.e., conjugate of GSH and epoxystearic acid, was isolated and characterized. The Michaelis constant (Km) values of the alpha, mu, and pi classes of GSTs for 9,10-epoxystearic acid were found to be 0.47, 0.32 and 0.80 mM, respectively, whereas the maximal velocity (V max) values for the alpha, mu, and pi classes of GSTs were found to be 142, 256, and 52 mol/min/mol, respectively. These results indicate that even though 9,10-epoxystearic acid is a substrate for all the three classes of GSTs, the mu class isozymes have maximum activity toward this substrate and may preferentially metabolize fatty acid epoxides more effectively as compared to the other classes of GSTs.     

Conjugation of isoprene monoepoxides with glutathione, catalyzed by alpha, mu, pi and theta-class glutathione S-transferases of rat and man

In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the alpha, mu, pi and theta-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the mu and theta class glutathione S-transferases were much more efficient than the alpha and pi class glutathione S-transferases, both for rat and man. Because the mu- and theta-class glutathione S-transferases are expressed in about 50 and 40-90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glu tathione S-transferases: rat GST T1-1 showed about 2.1-6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2-14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50-2000 microM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25-50-fold lower rate of GSH conjugation.     

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.     

Immunohistochemical localization, purification, and characterization of human urinary bladder glutathione S-transferases

This study describes immunohistochemical localization, purification and characterization of glutathione S-transferase (GST) of human urinary bladder. Even though all the three major classes of isoenzymes (alpha, mu, and pi) were expressed in human bladder, more than 90% of total GST activity was accounted for by a pi class anionic form. Human bladder alpha, mu, and pi class GSTs were immunologically related to respective isoenzymes of other human tissues. GST pi was present in all 13 samples analyzed, whereas GST alpha and mu were detected in nine and eleven samples, respectively. GST alpha of human bladder appeared to be unique, because unlike this class of GSTs of other human tissues, bladder enzyme had lower affinity for GSH linked to epoxy-activated Sepharose 6B affinity resin. Immunohistochemical staining indicated localization of GST alpha in epithelial surface cells, underlying submucosa and smooth muscle, whereas mu and pi class isoenzymes were predominantly distributed in epithelial surface cells. These results suggest that human bladder GSTs may play an important role in providing protection against xenobiotics because epithelium is considered a target for several carcinogens and all the three classes of isoenzymes are expressed in these cells.     

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV.

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.     

Activation of rat glutathione transferases in class mu by active oxygen species

The activities of rat glutathione transferases (GSTs) 3-3, 3-4, 4-4 in Class mu towards 1-chloro-2,4-dinitrobenzene (CDNB) but not 1,2-dichloro-4-nitrobenzene were increased up to 5-fold during preincubation with 0.4 mM xanthine and xanthine oxidase in 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA. The activated GST 3-4, purified by S-hexylglutathione affinity chromatography after the treatment, had a higher specific activity (130 units/mg) than that of the nontreated (35 units/mg), the Km and Vmax values for glutathione or CDNB also were increased. Other rat GSTs in Class alpha and pi were inactivated by the same treatment. In the presence of superoxide dismutase, the activation of GST 3-4 did not occur.     

2,4-Dichlorophenoxyacetic Acid and Related Chlorinated Compounds Inhibit Two Auxin-Regulated Type-III Tobacco Glutathione S-Transferases

Two auxin-inducible glutathione S-transferase (GST, EC 2.5.1.18) isozymes from tobacco (Nicotiana tabacum, White Burley) were partially characterized. GST1-1 and GST2-1 are members of a recently identified new type of plant GST isozymes that we will here refer to as type III. Both enzymes were active, with 1-chloro-2,4-dinitrobenzene as a substrate, when expressed in bacteria as fusion proteins. The apparent Km for 1-chloro-2,4-dinitrobenzene was found to be 0.85 [plus or minus] 0.25 mM for GST1-1 and 0.20 [plus or minus] 0.15 mM for GST2-1. The apparent Km for glutathione was similar for both enzymes, 0.40 [plus or minus] 0.15 mM. The in vitro activity of both enzymes could be inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic acid, with an apparent Ki of 80 [plus or minus] 40 [mu]M for GST1-1 and 200 [plus or minus] 100 [mu]M for GST2-1. The GST1-1 was also inhibited by structurally related substances, such as 2,4-dichlorobenzoic acid, with a roughly similar Ki. The nonchlorinated structures benzoic acid and phenoxyacetic acid did not inhibit. p-Chloroisobutyric acid, or clofibric acid, an auxin-transport inhibitor, was found to be an active inhibitor as well. The strongest inhibitor identified, however, was a phenylacetic acid derivative, ethacrynic acid, which showed an apparent Ki of 5 [plus or minus] 5 [mu]M for both enzymes. This substance is a known inducer as well as a substrate of specific mammalian GSTs. The results presented here indicate that the type III plant GSTs might be involved in the metabolism or transport of chlorinated substances that are structurally related to auxins. The possibility that auxins are endogenous ligands or substrates for GSTs is discussed.     

Amino acid sequence of glutathione S-transferase b from guinea pig liver

The amino acid sequence of glutathione S-transferase b (GST b) from guinea pig liver was determined by conventional methods. GST b was composed of two identical subunits, each with 217 amino acid residues. As GSTs are generally classified into three classes, alpha, mu, and pi, GST b belonged to class mu and the amino acid sequence of GST b showed about 80% homology with that of rat GST Yb.     

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.     

Glutathione S-transferases in normal and malignant human colon tissue

This study focuses on the GST composition of a tissue intrinsically resistant to chemotherapy, the human colon. GSTs were purified from matched pairs of colon tissue (normal and tumor) using glutathione affinity chromatography. The mean GST activity of colon tumors was 1.5-fold higher than that of normal tissue, with tumors of the sigmoid colon showing the greatest increase (2.3-fold). Two-dimensional gel electrophoresis and Western blot analysis of purified enzymes demonstrated the presence of all three GST classes (alpha, mu and pi) in colon, with GST pi being both the predominant isozyme in normal and malignant tissues. The level of alpha class subunits was the same in normal and tumor tissues, while the mu class subunits were decreased in tumors. A protein copurifying with GSTs from both normal and tumor tissue did not crossreact with GST antibodies, but instead reacted with a polyclonal antibody to glyoxylase I. This enzyme existed as a dimer in its native state. Upon boiling, monomeric subunits were produced with a molecular mass of 22.6 kDa and an isoelectric point more acidic than GST pi. Increased amounts of glyoxylase I were also found in tumor vs. normal colon. The apparent elevated levels of these glutathione-associated detoxifying enzymes in colon tumors may contribute to their intrinsic drug resistance.     

Effects of extract of Ginkgo biloba leaves and its constituents on carcinogen-metabolizing enzyme activities and glutathione levels in mouse liver

The effects of a standardized extract of Ginkgo biloba L. leaves (EGb) and its terpene constituents, bilobalide and ginkgolides, on the activities of detoxification enzymes, i.e., glutathione S-transferases (GSTs) and DT-diaphorase, and glutathione contents, were investigated in the mouse liver. Oral treatment with EGb (100-1,000 mg/kg) and bilobalide (10-30 mg/kg) once a day for 4 days caused a dose-dependent elevation in GST activity. Ginkgolide A (30 mg/kg, for 4 days) also significantly elevated GST activity, whereas ginkgolide B and ginkgolide C at the same dose had no effects. EGb significantly increased the protein level of GST pi, and bilobalide significantly increased those of GST alpha and GST mu Moreover, EGb-treatment and bilobalide-treatment caused significant elevations in DT-diaphorase activity and in hepatic glutathione contents.     

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.     

Bivalent inhibitors of glutathione S-transferase: the effect of spacer length on isozyme selectivity

Glutathione S-transferases (GSTs) are cytosolic enzymes that catalyze the conjugation of glutathione with a variety of exogenous and endogenous electrophiles. High affinity, isozyme-specific inhibitors of GST are required for use as pharmacological tools as well as potential therapeutics. The design of selective inhibitors is hindered due to the broad substrate binding capabilities of the GST enzymes. GSTs are dimeric enzymes, and therefore offer a unique discriminator for achieving inhibitor selectivity: the distance between binding sites on each monomer unit as a function of its quaternary organization. Bivalent analogs of the non-selective GST inhibitor ethacrynic acid were prepared, and selectivity for the GST A1-1 isozyme over GST P1-1 (IC50 values of 13.7 vs 1022 nM, respectively) was achieved through the optimization of the spacer length between the ethacrynic acid ligand domains.     

Do the major human glutathione S-transferases have fatty acid ethyl ester synthase activity?

Two fatty acid ethyl ester (FAEE) synthase isoenzymes purified from human myocardium were reported to be glutathione S-transferases (GST) [(1989) Proc. Natl. Acad. Sci. USA 86, 4470-4473; and (1989) J. Clin. Invest. 84, 1942-1946]. In the present study, the FAEE synthase activity of several purified and well characterized human GSTs were examined with ethanol and [14C]oleic acid as substrates. Three isoenzymes, GST1, GST2 and GST3 which are members of the evolutionary classes mu, alpha, and pi, respectively, were studied and failed to show any significant synthesis of FAEE after 45 min incubation at 37 degrees C. FAEE synthase activity and GST3 activity in human placental extracts can be readily separated by ion exchange chromatography on DEAE cellulose. Thus the results show that FAEE synthase activity is not a feature of the major GSTs found in human tissues. The two FAEE synthase isoenzymes isolated by Bora et al. may have been co-purified with GST isoenzymes or these FAEE synthases may be members of the GST super family that have low specific activity in conventional GST assays and have not been previously described.     

Screening for recombinant glutathione transferases active with monochlorobimane

A rapid and facile colony assay has been developed for catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferases (GST), expressed in Escherichia coli. The basis of the method is the conjugation of glutathione (GSH) with the fluorogenic substrate monochlorobimane (MCB). This screening method makes it possible to isolate and characterize one recombinant clone that is active with MCB among thousands of inactive variants. Colonies containing GSTs that catalyze the conjugation of GSH with MCB display fluorescence under long-wavelength UV light. The fluorescence is visible instantly. One rat and 11 human GSTs representing four distinct enzyme classes were studied, and all except human GST T1-1 gave rise to fluorescent colonies. The colony assay based on MCB can consequently be broadly applied for identifying active GSTs both after subcloning of wild-type enzymes and in the screening of mutant libraries. Populations of bacteria expressing GSTs can also be analyzed by flow cytometry.     

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.     

Energy-dependent export of the 13-oxooctadecadienoic acid-glutathione conjugate from HT-29 cells and plasma membrane vesicles

Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, beta,gamma-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K(m) of 325.5 microM and a V(max) of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K(m) of 169 microM and a V(max) of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.