Alpha-class Glutathione S-transferase Research Articles

Characterization of a class alpha glutathione- S-transferase with glutathione peroxidase activity in human liver microsomes

A 25.5 kDa class alpha glutathione S-transferase (GST) designated as microsomal Ya-GST or M-GSTA has been purified to electrophoretic homogeneity from human liver microsomes. Limited proteolysis, gel filtration chromatography followed by EDTA, and alkaline Na 2CO 3 treatments of microsomes indicate that the M-GSTA is intrinsic to the microsomes. Western immunoblot analysis revealed that human liver M-GSTA and the previously reported 17-kDa microsomal GST (FEBS Lett. 315 (1993) 77) did not have immunological cross reactivity. The enzyme showed conjugation activity towards substrates like 1-chloro-2,4-nitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 4-hydroxy-2-nonenal (4-HNE), a genotoxic α,β-unsaturated aldehyde product of lipid peroxidation. In addition, the M-GSTA exhibited significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides as well as phosphatidylcholine hydroperoxide, but not with H 2O 2. C-terminal amino acid sequence analysis revealed a high homology with the human liver cytosolic GST-A1 and A3 isozymes. Western immunoblot analyses of the microsomes prepared from human hepatoblastoma (HepG2) showed that the expression of this M-GSTA was induced upon treatment with such prooxidants as H 2O 2, suggesting that it may play an important role in the protection of cellular membranes from peroxidative damage.

Functional role of the lock and key motif at the subunit interface of glutathione transferase p1-1.

The glutathione transferases (GSTs) represent a superfamily of dimeric proteins. Each subunit has an active site, but there is no evidence for the existence of catalytically active monomers. The lock and key motif is responsible for a highly conserved hydrophobic interaction in the subunit interface of pi, mu, and alpha class glutathione transferases. The key residue, which is either Phe or Tyr (Tyr(50) in human GSTP1-1) in one subunit, is wedged into a hydrophobic pocket of the other subunit. To study how an essentially inactive subunit influences the activity of the neighboring subunit, we have generated the heterodimer composed of subunits from the fully active human wild-type GSTP1-1 and the nearly inactive mutant Y50A obtained by mutation of the key residue Tyr(50) to Ala. Although the key residue is located far from the catalytic center, the k(cat) value of mutant Y50A decreased about 1300-fold in comparison with the wild-type enzyme. The decrease of the k(cat) value of the heterodimer by about 27-fold rather than the expected 2-fold in comparison with the wild-type enzyme indicates that the two active sites of the dimeric enzyme work synergistically. Further evidence for cooperativity was found in the nonhyperbolic GSH saturation curves. A network of hydrogen-bonded water molecules, found in crystal structures of GSTP1-1, connects the two active sites and the main chain carbonyl group of Tyr(50), thereby offering a mechanism for communication between the two active sites. It is concluded that a subunit becomes catalytically competent by positioning the key residue of one subunit into the lock pocket of the other subunit, thereby stabilizing the loop following the helix alpha2, which interacts directly with GSH.

Endoplasmic reticulum stress due to altered cellular redox status positively regulates murine hepatic CYP2A5 expression.

Murine hepatic cytochrome P450 2A5 (CYP2A5) is uniquely induced by a variety of agents that cause liver injury and inflammation, conditions that are typically associated with downregulation of P450s. We hypothesized that induction of CYP2A5 occurs in response to hepatocellular damage resulting in endoplasmic reticulum (ER) stress. Treatment of mice in vivo and mouse hepatocytes in primary culture with the CYP2A5 inducer pyrazole resulted in overexpression of the ER stress biomarker glucose-regulated protein (GRP) 78. Treatment of primary hepatocytes with ER stress activators thapsigargin, tunicamycin, and trans-4,5-dihydroxy-1,2-dithiane (DTT(ox)) and the calcium ionophore A23187 (calcimycin) resulted in elevated GRP78 mRNA levels; however, only the reducing agent DTT(ox) induced levels of CYP2A5 mRNA, protein, and coumarin 7-hydroxylase activity. To test the hypothesis that CYP2A5 induction is due to liver injury resulting from altered cellular redox status, we demonstrated that CYP2A5 induction, elevated serum alanine aminotransferase, and oxidative protein damage occur concurrently in pyrazole-treated mice. Pyrazole also induced the expression of cytosolic alpha and mu class glutathione S-transferase expression both in vivo and in primary mouse hepatocytes. Moreover, treatment of hepatocytes with the redox cycling quinone menadione resulted in overexpression of CYP2A5 and GSTM1 mRNA. Finally, pretreatment of hepatocytes with the antioxidants N-acetylcysteine and vitamin E attenuated pyrazole-mediated increases in CYP2A5 mRNA levels. These findings clearly indicate that induction of mouse hepatic CYP2A5 during liver injury occurs via a novel mechanism involving ER stress due to altered cellular redox status.

Programmed delivery of novel functional groups to the alpha class glutathione transferases.

Here we describe a new route to site- and class-specific protein modification that will allow us to create novel functional proteins with artificial chemical groups. Glutathione transferases from the alpha but not the mu, pi, omega, or theta classes can be rapidly and site-specifically acylated with thioesters of glutathione (GS-thioesters) that are similar to compounds that have been demonstrated to occur in vivo. The human isoforms A1-1, A2-2, A3-3, and A4-4 from the alpha class all react with the reagent at a conserved tyrosine residue (Y9) that is crucial in catalysis of detoxication reactions. The yield of modified protein is virtually quantitative in less than 30 min under optimized conditions. The acylated product is stable for more than 24 h at pH 7 and 25 degrees C. The modification is reversible in the presence of excess glutathione, but the labeled protein can be protected by adding S-methylglutathione. The stability of the ester with respect to added glutathione depends on the acyl moiety. The reaction can also take place in Escherichia coli lysates doped with alpha class glutathione transferases. A control substance that lacks the peptidyl backbone required for binding to the glutathione transferases acylates surface-exposed lysines. There is some acyl group specificity since one out of the three different GS-thioesters that we tried was not able to acylate Y9.

Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal, a key molecule in stress mediated signaling.

Role of lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE) in cell cycle signaling is becoming increasingly clear. In this article, recent studies suggesting an important role of 4-HNE in stress mediated signaling for apoptosis are critically evaluated. Evidence demonstrating the modulation of UV, oxidative stress, and chemical stress mediated apoptosis by blocking lipid peroxidation by the alpha-class glutathione S-transferases (GSTs) is presented which suggest an important role of these enzymes in protection against oxidative stress and a role of lipid peroxidation products in stress mediated signaling. Overexpression of 4-HNE metabolizing GSTs (mGSTA4-4, hGSTA4-4, or hGST5.8) protects cells against 4-HNE, oxidative stress (H(2)O(2) or xanthine/xanthine oxidase), and UV-A mediated apoptosis by blocking JNK and caspase activation suggesting a role of 4-HNE in the mechanisms of apoptosis caused by these stress factors. The intracellular concentration of 4-HNE appears to be crucial for the nature of cell cycle signaling and may be a determinant for the signaling for differentiation, proliferation, transformation, or apoptosis. The intracellular concentrations of 4-HNE are regulated through a coordinated action of GSTs (GSTA4-4 and hGST5.8) which conjugate 4-HNE to GSH to form the conjugate (GS-HNE) and the transporter 76 kDa Ral-binding GTPase activating protein (RLIP76), which catalyze ATP-dependent transport of GS-HNE. A mild stress caused by heat, UV-A, or H(2)O(2)with no apparent effect on the cells in culture causes a rapid, transient induction of hGST5.8 and RLIP76. These stress preconditioned cells acquire ability to metabolize and exclude 4-HNE at an accelerated pace and acquire relative resistance to apoptosis by UV and oxidative stress as compared to unconditioned control cells. This resistance of stress preconditioned cells can be abrogated by coating the cells with anti-RLIP76 antibodies which block the transport of GS-HNE. These studies and previous reports discussed in this article strongly suggest a key role of 4-HNE in stress mediated signaling.

Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal, a key molecule in stress mediated signaling.

Role of lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE) in cell cycle signaling is becoming increasingly clear. In this article, recent studies suggesting an important role of 4-HNE in stress mediated signaling for apoptosis are critically evaluated. Evidence demonstrating the modulation of UV, oxidative stress, and chemical stress mediated apoptosis by blocking lipid peroxidation by the alpha-class glutathione S-transferases (GSTs) is presented which suggest an important role of these enzymes in protection against oxidative stress and a role of lipid peroxidation products in stress mediated signaling. Overexpression of 4-HNE metabolizing GSTs (mGSTA4-4, hGSTA4-4, or hGST5.8) protects cells against 4-HNE, oxidative stress (H(2)O(2) or xanthine/xanthine oxidase), and UV-A mediated apoptosis by blocking JNK and caspase activation suggesting a role of 4-HNE in the mechanisms of apoptosis caused by these stress factors. The intracellular concentration of 4-HNE appears to be crucial for the nature of cell cycle signaling and may be a determinant for the signaling for differentiation, proliferation, transformation, or apoptosis. The intracellular concentrations of 4-HNE are regulated through a coordinated action of GSTs (GSTA4-4 and hGST5.8) which conjugate 4-HNE to GSH to form the conjugate (GS-HNE) and the transporter 76 kDa Ral-binding GTPase activating protein (RLIP76), which catalyze ATP-dependent transport of GS-HNE. A mild stress caused by heat, UV-A, or H(2)O(2)with no apparent effect on the cells in culture causes a rapid, transient induction of hGST5.8 and RLIP76. These stress preconditioned cells acquire ability to metabolize and exclude 4-HNE at an accelerated pace and acquire relative resistance to apoptosis by UV and oxidative stress as compared to unconditioned control cells. This resistance of stress preconditioned cells can be abrogated by coating the cells with anti-RLIP76 antibodies which block the transport of GS-HNE. These studies and previous reports discussed in this article strongly suggest a key role of 4-HNE in stress mediated signaling.

The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1

The thioredoxin-like fold has a betaalphabetaalphabetabetaalpha topology, and most proteins/domains with this fold have a topologically conserved cis -proline residue at the N-terminus of beta-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347-350]. In order to further address the role of the cis -proline in the structure, function and stability of GSTs, cis -Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.

Evaluation of 2-year-old intrasplenic fetal liver tissue transplants in rats.

Liver cell transplantation into host organs like the spleen may possibly provide a temporary relief after extensive liver resection or severe liver disease or may enable treatment of an enzyme deficiency. With time, however, dedifferentiation or malignant transformation of the ectopically transplanted cells may be possible. Thus, in the present study syngenic fetal liver tissue suspensions were transplanted into the spleen of adult male rats and evaluated 2 years thereafter in comparison to orthotopic livers for histopathological changes and (as markers for preneoplastic transformation) for cytochrome P450 (P450) and glutathione S-transferase (GST) isoform expression. Because inducibility of P450 and GST isoforms may be changed in preneoplastic foci, prior to sacrifice animals were additionally treated either with beta-naphthoflavone, phenobarbital, dexamethasone, or the respective solvent. In the 2-year-old grafts more than 70% of the spleen mass was occupied by the transplant. The transplanted hepatocytes were arranged in cord-like structures. Also few bile ducts were present. Morphologically, no signs of malignancy were visible. With all rats, transplant recipients as well as controls, however, discrete nodular structures were seen in the livers. Due to age, both livers and transplants displayed only a low P450 2B1 and 3A2 and GST class alpha and mu isoform expression. No immunostaining for P450 1A1 was visible. At both sites, beta-naphthoflavone, phenobarbital, or dexamethasone treatment enhanced P450 1A1, P450 2B1 and 3A2, or P450 3A2 expression, respectively. No immunostaining for GST class pi isoforms was seen in the transplants. The livers of both transplant recipients and control rats, however, displayed GST pi-positive foci, corresponding to the nodular structures seen histomorphologically. Compared to the surrounding tissue, these foci also exhibited a more pronounced staining for GST class alpha and mu isoforms and a stronger inducibility of the P450 1A1 expression due to beta-naphthoflavone. In conclusion, in contrast to the livers, no preneoplastic foci seem to appear in the intrasplenic transplants even 2 years after transplantation. This may be due either to the protection of these transplants by the orthotopic livers or to the different humoral and nerval influences at the ectopic site.

Insulin and glucagon regulation of glutathione S-transferase expression in primary cultured rat hepatocytes.

Diabetes is a major cause of morbidity and mortality, and complications resulting from diabetes have been attributed in part to increased oxidative stress. Glutathione S-transferases (GSTs) constitute a major protective mechanism against oxidative stress. Studies of the expression and activity of GSTs during diabetes are inconclusive, with both increased and decreased GST expression being reported in vivo. Insulin and glucagon effects on GST expression and the signaling pathway involved in the glucagon regulation of GST expression were examined in primary cultured rat hepatocytes. The addition of insulin resulted in the elevation of alpha-class GST protein levels, whereas alpha- and pi-class GST protein levels were markedly decreased in hepatocytes cultured with glucagon. In contrast, mu-class GST protein expression was unaffected by insulin or glucagon treatment. Insulin concentrations >/=1 nM resulted in increased GST activities and alpha-class GST protein levels, whereas glucagon concentrations >/=20 nM decreased alpha- and pi-class protein levels and activity. Treatment of cells with 8-bromo-cAMP or dibutyryl-cAMP also resulted in decreased alpha- and pi-class GST protein levels. Pretreatment with N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89), a selective inhibitor of protein kinase A, before glucagon addition markedly attenuated the glucagon effect. This study demonstrates that insulin and glucagon regulate, in an opposing manner, the expression of alpha-class GSTs and that glucagon completely inhibits pi-class GST expression in vitro, suggesting that hepatic GST expression may be decreased during diabetes. Furthermore, the present study implicates cAMP and protein kinase A in mediating the inhibitory effect of glucagon on GST expression.

Residues 207, 216, and 221 and the catalytic activity of mGSTA1-1 and mGSTA2-2 toward benzo[a]pyrene-(7R,8S)-diol-(9S,10R)-epoxide.

Murine class alpha glutathione S-transferase subunit types A2 (mGSTA2-2) and A1 (mGSTA1-1) have high catalytic efficiency for glutathione (GSH) conjugation of the ultimate carcinogenic metabolite of benzo[a]pyrene, (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene, [(+)-anti-BPDE]. Only 10 residues differ between the sequences of mGSTA1-1 and 2-2. However, the catalytic efficiency of mGSTA1-1 for GSH conjugation of (+)-anti-BPDE is >3-fold higher as compared with mGSTA2-2. The crystal structure of mGSTA1-1 in complex with the GSH conjugate of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (GSBpd) reveals that R216 and I221 in the last helix play important roles in catalysis [Gu, Y., Singh, S. V., and Ji, X. (2000) Biochemistry 39, 12552-12557]. The crystal structure of mGSTA2-2 in complex with GSBpd has been determined, which reveals a different binding mode of GSBpd. Comparison of the two structures suggests that residues 207 and 221 are responsible for the different binding mode of GSBpd and therefore contribute to the distinct catalytic efficiency of the two isozymes.

Developmental changes in glutathione S-transferase isoforms expression and activity in intrasplenic fetal liver tissue transplants in rats

The aim of the present study was to characterise developmental changes in glutathione S-transferase (GST) isoforms expression and in glutathione conjugation capacity in intrasplenic liver tissue transplants. For this purpose, syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fischer 344 rats. Three days, 1, 2, 4 weeks, 2, 4, 6 months and 1 year later, transplant-recipients and control animals were sacrificed and class alpha, mu and pi GST isoforms expression and GST activities using the substrates o-dinitrobenzene and 1-chloro-2,4-dinitrobenzene were assessed in livers and spleens. In the hepatocytes of the adult livers no class pi, but a distinct class alpha and mu GST expression was seen. The bile duct epithelia were class pi GST positive. Fetal livers displayed almost no class alpha and mu, but a slight class pi GST expression. The same pattern was seen in 3-day-old intrasplenic liver tissue transplants. Up to 2 weeks after surgery the class alpha and mu GST expression increased in the hepatocytes of the transplants, whereas the immunostaining for class pi GST disappeared. No remarkable changes were seen thereafter. Normal conjugation capacities were observed with the livers of both groups of rats. Control spleens displayed only low GST activities. From 2 months after transplantation on activities were significantly higher in transplant-containing spleens than in respective control organs with a further increase up to one year after grafting. These results show that intrasplenically transplanted fetal liver cells proliferate and differentiate into mature cells displaying a GST expression pattern with respective enzyme activities similar to adult liver.

Down-regulation of alpha class glutathione S-transferase by interleukin-1beta in human intestinal epithelial cells (Caco-2) in culture.

The influence of pro-inflammatory cytokines on alpha class glutathione S-transferase A1 and A2 (GSTA1/A2) expression was examined in human colonic epithelial cells (Caco-2) in culture. Dose-dependent reductions in GSTA1/A2 mRNA, protein, and activity levels occurred in Caco-2 cells cultured in conditioned medium (CM) from lipopolysaccharide-stimulated murine monocyte-macrophage cells (RAW 264.7). Neutralizing anti-interleukin-1beta (IL-1beta) antibodies attenuated this repression of GSTA1/A2 expression by CM. Moreover, recombinant human IL-1beta reduced GSTalpha expression at the mRNA, protein, and activity levels in a dose-related fashion. Reduction of GSTA1/A2 mRNA levels by IL-1beta was attenuated by pretreatment with IL-1 receptor antagonist. GSTA1/A2 mRNA half-lives were similar in control and IL-1beta-treated cells, indicating that IL-1beta has no effect on mRNA stability. In reporter gene studies, IL-1beta caused a dose-related reduction of luciferase activity in Caco-2 cells transfected with the full-length GSTA1 promoter-luciferase construct. Using truncated constructs, IL-1beta responsiveness was mapped to a region 286 base pairs upstream to the coding region. Deletion of a hepatic nuclear factor 1 (HNF-1) site in this region abrogated the IL-1beta-mediated repression of GSTA1 promoter activity. These results demonstrate that IL-1beta down-regulates GSTA1/A2 expression in cultured human enterocytes by a transcriptional mechanism involving an HNF-1 site.

Conjugation of 4-hydroxynonenal by largemouth bass (Micropterus salmoides) glutathione S-transferases

The glutathione S-transferases (GST) are a major group of conjugative enzymes involved in the detoxification of electrophilic compounds and products of oxidative stress. We have previously described the kinetics of hepatic GST conjugation in largemouth bass using a variety of synthetic GST reference substrates. In the present study, we investigated the ability of largemouth bass hepatic GSTs to conjugate 4-hydroxynon-2-enal (4HNE), a mutagenic and cytotoxic α-β-unsaturated aldehyde produced during oxidative injury. Hepatic cytosolic fractions from largemouth bass rapidly catalyzed GSH-dependent 4HNE conjugation, with the rate of GST-4HNE conjugation in bass liver exceeding those of several other mammalian and aquatic species. No apparent sex-related differences in GST-4HNE activity were observed among adult bass. SDS-PAGE and Western blotting analysis of GSH affinity-purified bass liver cytosolic GST revealed the presence of two major GST subunits of approximately 30 and 27 KDa that exhibited slight cross-reactivity when probed with a rat alpha class GST antibody, but not to rat mu, pi or theta class GST. The rapid conjugation of 4HNE by hepatic GST suggests an important role for GSTs in protecting against peroxidation of polyunsaturated fatty acids in bass liver.

Comparative expression of two alpha class glutathione S-transferases in human adult and prenatal liver tissues

The ability of the fetus to detoxify transplacental drugs and chemicals can be a critical determinant of teratogenesis and developmental toxicity. Developmentally regulated expression of alpha class glutathione S-transferases (GSTs) is of particular interest, since these isozymes have high activity toward peroxidative byproducts of oxidative injury that are linked to teratogenesis. The present study was initiated to examine the expression and catalytic activities of alpha class GST isozymes in human prenatal liver. Northern analysis demonstrated the presence of hGSTA1 and/or A2 (hGSTA1/2) and hGSTA4 steady-state mRNAs in second trimester prenatal livers. Western blotting of prenatal liver proteins provided corroborating evidence via detection of an hGSTA1/2-reactive protein in both cytosol and mitochondria and of hGSTA4-4-reactive protein in mitochondria alone. Catalytic studies demonstrated that prenatal liver cytosolic GSTs were active toward 1-chloro-2,4-dinitrobenzene (a general GST reference substrate), δ 5-androstene-3,17-dione (relatively specific for hGSTA1-1), and 4-hydroxynonenal, a highly mutagenic α,β-unsaturated aldehyde produced during oxidative damage and a substrate for hGSTA4-4. Total GSH-peroxidase and GST-dependent peroxidase activities were 9- and 18-fold higher, respectively, in adult liver than in prenatal liver. Multiple tissue array analyses demonstrated considerable tissue-specific and developmental variation in GST mRNA expression. In summary, our results demonstrate the presence of two important alpha class GSTs in second trimester human prenatal tissues, and indicate that mitochondrial targeting of GST may represent an important pathway for removal of cytotoxic products in prenatal liver. Furthermore, the relatively inefficient prenatal reduction of hydroperoxides may underlie an increased susceptibility to maternally transferred pro-oxidant drugs and chemicals.

Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding

In addition to their catalytic functions, cytosolic glutathioneS-transferases (GSTs) are a major reserve of high-capacity binding proteins for a large variety of physiological and exogenous non-substrate compounds. This ligandin function has implicated GSTs in numerous ligand-uptake, -transport and -storage processes. The binding of non-substrate ligands to GSTs can inhibit catalysis. In the present study, the energetics of the binding of the non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to wild-type human class Alpha GST with two type-1 subunits (hGSTA1-1) and its ΔPhe-222 deletion mutant were studied by isothermal titration calorimetry. The stoichiometry of binding to both proteins is one ANS molecule per GST subunit with a greater affinity for the wild-type (Kd=65μM) than for the ΔPhe-222 mutant (Kd=105μM). ANS binding to the wild-type protein is enthalpically driven and it is characterized by a large negative heat-capacity change, ΔCp. The negative ΔCp value for ANS binding indicates a specific interface with a significant hydrophobic component in the protein—ligand complex. The negatively charged sulphonate group of the anionic ligand is apparently not a major determinant of its binding. Phe-222 contributes to the binding affinity for ANS and the hydrophobicity of the binding site.

Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding.

In addition to their catalytic functions, cytosolic glutathioneS-transferases (GSTs) are a major reserve of high-capacity binding proteins for a large variety of physiological and exogenous non-substrate compounds. This ligandin function has implicated GSTs in numerous ligand-uptake, -transport and -storage processes. The binding of non-substrate ligands to GSTs can inhibit catalysis. In the present study, the energetics of the binding of the non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to wild-type human class Alpha GST with two type-1 subunits (hGSTA1-1) and its DeltaPhe-222 deletion mutant were studied by isothermal titration calorimetry. The stoichiometry of binding to both proteins is one ANS molecule per GST subunit with a greater affinity for the wild-type (K(d)=65 microM) than for the DeltaPhe-222 mutant (K(d)=105 microM). ANS binding to the wild-type protein is enthalpically driven and it is characterized by a large negative heat-capacity change, DeltaC(p). The negative DeltaC(p) value for ANS binding indicates a specific interface with a significant hydrophobic component in the protein-ligand complex. The negatively charged sulphonate group of the anionic ligand is apparently not a major determinant of its binding. Phe-222 contributes to the binding affinity for ANS and the hydrophobicity of the binding site.

Inactivation of Carcinogenic Diol Epoxides of Dibenzo[ a,l ]pyrene (Dibenzo[ def,p ]chrysene) by Human Alpha Class Glutathione Transferases

Inactivation of Carcinogenic Diol Epoxides of Dibenzo[ a,l ]pyrene (Dibenzo[ def,p ]chrysene) by Human Alpha Class Glutathione Transferases

Inactivation of Carcinogenic Diol Epoxides of Dibenzo[ a,l ]pyrene (Dibenzo[ def,p ]chrysene) by Human Alpha Class Glutathione Transferases

Human Alpha class glutathione transferases (hGSTs) have been incubated with the ultimate carcinogenic ( m )- anti - and (+)- syn -diol epoxides (DE) of the nonplanar dibenzo[ a,l ]pyrene (DBP). hGSTA1-1, A2-2, and A3-3 demonstrate activity with both diol epoxides ( R -absolute configuration at the benzylic oxirane carbon) whereas hGSTA4-4 virtually was inactive. (+)- syn -DBPDE was superior as substrate compared to the ( m )- anti enantiomer with hGSTA1-1 as the most efficient enzyme (k cat /K M = 464 mM m 1 s m 1 ) followed by A3-3 and A2-2 (k cat /K M = 190 mM m 1 s m 1 and 30.4 mM m 1 s m 1 , respectively). With ( m )- anti -DBPDE, the k cat /K M values were in general about 10-fold lower. Replacing ( m )- anti -DBPDE with the less complex ( m )- anti -BCDE or the less structurally distorted (+)- anti -BPDE, revealed that GSTA1-1 was 19- and 25-fold less active, respectively. hGSTA1-1 is present in human lung, a primary target tissue for PAH-induced tumors. Considering the great efficiency of this isoform relative to both Pi and Mu-class GSTs toward DBPDE, its presence and extent of expression may play a significant role in protection against this type of highly carcinogenic compounds.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization

A 25 kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na(2)CO(3), indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73-83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87-101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization

A 25kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na2CO3, indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73–83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243–248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87–101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H2O2. Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.