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PubMed1992

An evolutionary perspective on glutathione transferases inferred from class-theta glutathione transferase cDNA sequences.

We report the cDNA sequence for rat glutathione transferase (GST) subunit 5, which is one of at least three class Theta subunits in this species. This sequence, when compared with that of subunit 12 recently published by Ogura, Nishiyama, Okada, Kajita, Narihata, Watabe, Hiratsuka & Watabe [(1991) Biochem. Biophys. Res. Commun. 181, 1294-1300] proves that Theta is a separate multigene class of GST with little amino acid sequence identity with Mu-, Alpha- or Pi-class enzymes. The amino acid sequence identity of class-Theta subunits is highly conserved in rat, the fruitfly Drosophila, maize (Zea mays) and Methylobacterium, which suggests that this family is representative of the ancient progenitor GST gene and originates from the endosymbioses of a purple bacterium leading to the mitochondrion. The high conservation of class Theta brings into prominence that Alpha-, Mu- and Pi-class enzymes, which are not present in plants, derive from a Theta-class gene duplication before the divergence of fungi and animals and, given the binding properties of the Alpha-, Mu- and Pi-classes, suggests a role for these in the evolution of fungi and animals.

Amino Acid Sequence↗
PubMed1994

Co-variation of glutathione transferase expression and cytostatic drug resistance in HeLa cells: establishment of class Mu glutathione transferase M3-3 as the dominating isoenzyme.

Qualitative and quantitative analyses of glutathione, glutathione transferases (GSTs) and other glutathione-linked enzymes in HeLa cells have been made in order to study their significance in cellular resistance to electrophilic cytotoxic agents. The cytosolic concentrations of three GSTs, GST M1-1 (53 +/- 9 ng/mg of cytosolic protein), GST P1-1 (11 +/- 3 ng/mg) and GST A1-1 (1.1 +/- 0.4 ng/mg) were quantified by isoenzyme-specific enzyme-linked immunoassays. Electrophoretic analysis and immunoblotting demonstrated another component, GST M3-3, which was identified by amino acid sequence analysis. GST M3-3 was quantified (1550 +/- 250 ng/mg) by slot-blot immunoanalysis and was the most abundant GST in HeLa cells. An additional cytosolic 13 kDa protein with high affinity for immobilized glutathione or S-hexyglutathione was found to be identical with a macrophage migration-inhibitory factor, previously identified as a lymphokine. Cells grown in roller bottles (HR) rather than in ordinary culture flasks contain a significantly lower concentration of all the GSTs and were found to be more sensitive to the cytostatic agents doxorubicin (2.3-fold), cisplatin (1.7-fold) and melphalan (1.4-fold). The cytosolic concentrations of glutathione reductase and glyoxalase I were also lower in HR cells, whereas the total glutathione concentration was unchanged and the glutathione peroxidase activity was increased. The results indicate that GSTs contribute to the cellular resistance phenotype.

Amino Acid Sequence↗
PubMed1994

Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.

Affinity Labels↗
PubMed1997

Cloning and overexpression of rat kidney biliverdin IX alpha reductase as a fusion protein with glutathione S-transferase: stereochemistry of NADH oxidation and evidence that the presence of the glutathione S-transferase domain does not effect BVR-A activity.

Native biliverdin IX alpha reductase (BVR-A) is a monomer of molecular mass 34 kDa. We have developed an expression vector that allows the isolation of 40 mg of a glutathione S-transferase (GST)-BVR-A fusion protein from 1 litre of culture. The fusion protein (60 kDa) behaves as a dimer on gel filtration (120 kDa), so that we have artificially created a BVR-A dimer. The recombinant rat kidney enzyme exhibits pre-steady-state 'burst' kinetics that show a pH dependence similar to that already described for ox kidney BVR-A. Similar behaviour was obtained in the presence and absence of the GST domain both for the burst kinetics and during initial-rate studies in the presence and absence of albumin. The stereospecificity of the BVR-A-catalysed oxidation of [4-3H]NADH, labelled at the A and B faces, was shown to occur exclusively via the B face.

Alcohol Dehydrogenase↗
PubMed1995

Site-directed mutagenesis of human glutathione transferase P1-1. Mutation of Cys-47 induces a positive cooperativity in glutathione transferase P1-1.

Glutathione transferase P1-1 (EC 2.5.1.18) is a dimeric enzyme composed of identical subunits each containing one binding site for GSH and a second for the co-substrate e.g. 1-chloro-2,4-dinitrobenzene. Steady-state kinetics are strictly hyperbolic toward both these substrates. Replacement of Cys-47 with alanine or serine decreases the affinity for GSH and triggers a positive kinetic cooperativity with respect to the substrate. Hill coefficients were 1.31 and 1.43 for the C47A and C47S mutants. C47A/C101S and C47S/C101S double mutants display lower affinity for GSH and higher Hill coefficients (1.57 and 1.56, respectively) when compared with C47A and C47S single mutants. Conversely, replacement of Cys-101 with alanine or serine does not yield any cooperativity and any marked change of kinetic parameters. Fluorometric experiments gave sigmoidal isothermic GSH binding curves for all the Cys-47 mutants, with Hill coefficients similar to that obtained by the kinetic approach. These data, together with the activation experiments performed in the presence of S-hexylglutathione, suggest that the substitution of Cys-47 yields a dimeric low-affinity enzyme which may be revealed by the lack of a peculiar electrostatic bond between the thiolate form of Cys-47 and the protonated amino group of Lys-54.

Cysteine↗
PubMed1996

Structural flexibility modulates the activity of human glutathione transferase P1-1. Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase.

Presteady-state and steady-state kinetics of human glutathione transferase P1-1 (EC 2.5.1.18) have been studied at pH 5.0 by using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a poor co-substrate for this isoenzyme. Steady-state kinetics fits well with the simplest rapid equilibrium random sequential bi-bi mechanism and reveals a strong intrasubunit synergistic modulation between the GSH-binding site (G-site) and the hydrophobic binding site for the co-substrate (H-site); the affinity of the G-site for GSH increases about 30 times at saturating co-substrate and vice versa. Presteady-state experiments and thermodynamic data indicate that the rate-limiting step is a physical event and, probably, a structural transition of the ternary complex. Similar to that observed with 1-chloro-2, 4-dinitrobenzene (Ricci, G., Caccuri, A. M., Lo Bello, M., Rosato, N. , Mei, G., Nicotra, M., Chiessi, E., Mazzetti, A. P., and Federici, G.(1996) J. Biol. Chem. 271, 16187-16192), this event may be related to the frequency of enzyme motions. The observed low, viscosity-independent kcat value suggests that these motions are slow and diffusion-independent for an increased internal viscosity. In fact, molecular modeling suggests that the hydroxyl group of Tyr-108, which resides in helix 4, may be in hydrogen bonding distance of the oxygen atom of this new substrate, thus yielding a less flexible H-site. This effect might be transmitted to the G-site via helix 4. In addition, a new homotropic behavior exhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole is found in Cys-47 mutants revealing a structural intersubunit communication between the two H-sites.

4-Chloro-7-nitrobenzofurazan↗
PubMed1997

Catalytic mechanism and role of hydroxyl residues in the active site of theta class glutathione S-transferases. Investigation of Ser-9 and Tyr-113 in a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina.

Spectroscopic and kinetic studies have been performed on the Australian sheep blowfly Lucilia cuprina glutathione S-transferase (Lucilia GST; EC 2.5.1.18) to clarify its catalytic mechanism. Steady state kinetics of Lucilia GST are non-Michaelian, but the quite hyperbolic isothermic binding of GSH suggests that a steady state random sequential Bi Bi mechanism is consistent with the anomalous kinetics observed. The rate-limiting step of the reaction is a viscosity-dependent physical event, and stopped-flow experiments indicate that product release is rate-limiting. Spectroscopic and kinetic data demonstrate that Lucilia GST is able to lower the pKa of the bound GSH from 9.0 to about 6.5. Based on crystallographic suggestions, the role of two hydroxyl residues, Ser-9 and Tyr-113, has been investigated. Removal of the hydroxyl group of Ser-9 by site-directed mutagenesis raises the pKa of bound GSH to about 7.6, and a very low turnover number (about 0.5% of that of wild type) is observed. This inactivation may be explained by a strong contribution of the Ser-9 hydroxyl group to the productive binding of GSH and by an involvement in the stabilization of the ionized GSH. This serine residue is highly conserved in the Theta class GSTs, so the present findings may be applicable to all of the family members. Tyr-113 appears not to be essential for the GSH activation. Stopped-flow data indicate that removal of the hydroxyl group of Tyr-113 does not change the rate-limiting step of reaction but causes an increase of the rate constants of both the formation and release of the GSH conjugate. Tyr-113 resides on alpha-helix 4, and its hydroxyl group hydrogen bonds directly to the hydroxyl of Tyr-105. This would reduce the flexibility of a protein region that contributes to the electrophilic substrate binding site; segmental motion of alpha-helix 4 possibly modulates different aspects of the catalytic mechanism of the Lucilia GST.

Animals↗
PubMedDate not supplied

Methylguanine DNA methyl transferase activities, glutathione s transferase and nitric oxide in bladder cancer patients.

Tumor formation is a multistep process that can be divided in to the stages of tumor initiation, promotion, and progression. DNA repair protein; MGMT is a key suicide enzyme that repairs the mispairing base methylguanine, which is induced in DNA as a minor lesion. The glutathione S transferases (GSTs) are a family of enzymes that are important to protect against alkylating agents. Nitric oxide, contributes to the regulation of tumor angiogenesis. A substantial body of experimental evidence supports the hypothesis that tumor angiogenesis is fundamental for the growth and metastasis of solid tumors. We measured the activities of GST, MGMT, and levels of NO3-/NO2- in the leukocytes from patients with bladder carcinoma and healthy controls and activities of MGMT in the tissue from patients with bladder carcinoma and adjacent normal tissue in bladder. Both GST and tissue MGMT activites were significantly increased in the patient group. There was no significant difference between controls and patients for MGMT activity in peripheral blood leukocytes (PBL). Nitrate/nitrite levels in PBL, there was no significant difference between controls and patients. Nitrate/nitrite levels were increased in G2-G3 tumors. In conclusion, we determined high concentrations of nitrite in leukocytes are suspected alkylation damage by induction nitrosamine. Increased DNA alkylation damage may lead the stimulation of MGMT and GST.

Aged↗
PubMed2002

Glutathione S-transferase and O6-methylguanine DNA methyl transferase activities in patients with thyroid papillary carcinoma.

Alkylating agents, which are metabolized by glutathione S-transferase (GST), have an important role in the etiology of cancer by forming mutagenic DNA adducts. Previous studies have shown that DNA repair protein, O6-methylguanine DNA methyltransferase, repairs these mutagenic DNA adducts and its activity is correlated with the resistance of human tumors to alkylating agent-based anti-cancer drugs. However, little is known about GST and O6-methylguanine DNA methyltransferase activities in patients with thyroid cancer in vivo. We measured the activities of GST and O6-methylguanine DNA methyltransferase in the leukocytes from patients with papillary thyroid carcinoma and healthy controls. The GST activity was significantly higher in men than in women, and it was negative correlated with age in men whereas it was unchanged in women in the control group. Both GST and O6-methylguanine DNA methyltransferase activities were significantly increased in the patient group. There were no age and sex-related changes in the O6-methylguanine DNA methyltransferase activity in both the control and patient groups. These results suggest that leukocyte GST and O6-methylguanine DNA methyltransferase activities were increased with thyroid cancer. This may be related to the resistance to chemotherapy exhibited by patients with thyroid cancer.

Adult↗
PubMed1985

Relative merits of immunohistochemical demonstrations of placental, A, B and C forms of glutathione S-transferase and histochemical demonstration of gamma-glutamyl transferase as markers of altered foci during liver carcinogenesis in rats.

The values of the immunohistochemical demonstrations of glutathione S-transferases (GSTs) A, B, C and P and histochemical demonstrations of gamma-glutamyl transpeptidase (gamma-GT) for detection of enzyme altered foci in F344 rat liver were compared. Rats were given a single i.p. injection of 200 mg/kg body weight of diethylnitrosamine (DENA), from 2 weeks later they were given 0.02% N-2-fluorenylacetamide (2-FAA), phenobarbital (PB), butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) in their diet for 6 weeks and then they were given basal diet and tap water for 4 weeks. They were subjected to partial hepatectomy at the end of week 3. Results showed that immunohistochemical demonstration of GSTs A, B and C for detection of foci were only effective when the administration of 2-FAA, PB, BHA or BHT in the diet was discontinued, because these GSTs were induced in surrounding hepatocytes by these compounds in the diet. gamma-GT was induced in periportal hepatocytes strongly by BHA and BHT and slightly by PB, and gamma-GT positive foci in periportal areas were not distinguishable from gamma-GT positive periportal hepatocytes. GST-P was also induced moderately by BHA and slightly by BHT in periportal hepatocytes, but all GST-P positive foci were clearly distinguishable. In addition, almost all gamma-GT positive foci gave a positive reaction for GST-P, but 5-10% of the GST-P positive foci were not gamma-GT positive.

Animals↗
PubMed1997

Partial characterization of glutathione S-transferases from wheat (Triticum spp.) and purification of a safener-induced glutathione S-transferase from Triticum tauschii.

Hexaploid wheat (Triticum aestivum L.) has very low constitutive glutathione S-transferase (GST) activity when assayed with the chloroacetamide herbicide dimethenamid as a substrate, which may account for its low tolerance to dimethenamid in the field. Treatment of seeds with the herbicide safener fluxofenim increased the total GST activity extracted from T. aestivum shoots 9-fold when assayed with dimethenamid as a substrate, but had no effect on glutathione levels. Total GST activity in crude protein extracts from T. aestivum, Triticum durum, and Triticum tauschii was separated into several component GST activities by anion-exchange fast-protein liquid chromatography. These activities (isozymes) differed with respect to their activities toward dimethenamid or 1-chloro-2,4-dinitrobenzene as substrates and in their levels of induction by safener treatment. A safener-induced GST isozyme was subsequently purified by anion-exchange and affinity chromatography from etiolated shoots of the diploid wheat species T. tauschii (a progenitor of hexaploid wheat) treated with the herbicide safener cloquintocet-mexyl. The isozyme bound to a dimethenamid-affinity column and had a subunit molecular mass of 26 kD based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme (designated GST TSI-1) was recognized by an antiserum raised against a mixture of maize (Zea mays) GSTs. Amino acid sequences obtained from protease-digested GST TSI-1 had significant homology with the safener-inducible maize GST V and two auxin-regulated tobacco (Nicotiana tabacum) GST isozymes.

Acetophenones↗
PubMed2006

Characterization of glutathione S-transferase from dust mite, Der p 8 and its immunoglobulin E cross-reactivity with cockroach glutathione S-transferase.

BACKGROUND: Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems. OBJECTIVE: To assess the allergenicity of native and recombinant mite glutathione S-transferase (GST) (Der p 8) and study the IgE cross-reactivity between Der p 8 and cockroach GST. METHODS: Der p 8 cDNA encoding a new isoform was isolated and expressed in yeast. Native Der p 8 was affinity purified from mite extract. IgE reactivity to native and recombinant Der p 8 was assessed by ELISA using sera from allergic subjects from Taiwan, Singapore and Malaysia. IgE cross-reactivity between Der p 8 and cockroach GST was examined by IgE inhibition assays. RESULTS: Our Der p 8 cDNA encoded a basic isoform (pI=8.5) containing six polymorphic residues located at positions 46, 106, 149, 160, 167 and 184. At least 8 isoforms of native Der p 8 were detected by two-dimensionalgel and immunoblot analyses. Sera from Taiwanese asthmatics showed 96% and 84% IgE reactivity to native Der p 8 and recombinant Der p 8, respectively. Native Der p 8 showed 75% and 65% IgE reactivity with sera from Malaysia and Singapore, respectively. CONCLUSIONS: A high frequency of sensitization to mite GST among allergic subjects was observed but the titres of IgE reactivity were low. The IgE cross-reactivity between mite and cockroach GST suggests that GST is a panallergen.

Adolescent↗
PubMed1986

Cytosolic rat liver glutathione transferase 4-4. Primary structure of the protein reveals extensive differences between homologous glutathione transferases of classes alpha and mu.

The primary structure of the class Mu glutathione transferase 4-4 from rat liver was determined. The structural data characterize a class Mu protein within an enzyme family for which three classes have been distinguished (Alpha, Mu, Pi). The structure was determined by analysis of peptides obtained after treatment with trypsin. Glu-specific protease and CNBr. The protein is composed of two identical subunits, each with 217 amino acid residues. No evidence for microheterogeneity or for the presence of modified residues was encountered. The primary structure was found to be strictly homologous with corresponding parts in known regions of other class Mu enzymes of rat, mouse, human and bovine origin. Relationships to the cytosolic enzyme of other classes (Pi and Alpha) are considerably more distant. A comparison with the entire chain of the class Alpha subunit 1 from rat liver was carried out by three methods, alignment of amino acid sequences, correlation of hydrophilicity plots and predictions of secondary structures. All methods reveal weak similarities but also large differences. The overall positional identity is only 26%. Combined, the results establish the first complete class Mu structure, show distant inter-class relationships, and relate subunit 4 (class Mu) and subunit 1 (class Alpha) in a family of enzymes rather than in a group of isoenzymes.

Amino Acid Sequence↗
PubMed1986

Mitochondrial glutathione transferases. The alkylation of mitochondrial membrane yields a catalyst with glutathione-transferase-like properties.

The pretreatment of rat liver mitochondria with alkylating agents (N-ethylmaleimide, iodoacetamide or vinyl pyridine) increased the activity of mitochondrial glutathione transferase (GST) by 100-250%. Further experiments provided evidence that mitochondrial membranes contain an enzymatically inactive protein which can be alkylated through its sulfhydryl groups. By alkylation, this protein achieves catalytic properties similar to those of native GST.

Alkylating Agents↗
PubMed2000

Normal bronchial epithelial cell expression of glutathione transferase P1, glutathione transferase M3, and glutathione peroxidase is low in subjects with bronchogenic carcinoma.

Normal bronchial epithelial cells (NBECs) are at risk for damage from inhaled and endogenous oxidative species and from epoxide metabolites of inhaled polycyclic aromatic hydrocarbons. Epidemiological and in vitro data suggest that interindividual variation in this risk may result from variation in NBEC expression of enzymes that inactivate reactive species by conjugating them to glutathione. Quantitative competitive reverse transcription-PCR was used to measure mRNA levels of glutathione transferases (GSTs) and glutathione peroxidases (GSHPxs) in primary NBECs from subjects with or without bronchogenic carcinoma. Mean expression levels (mRNA/10(3) beta-actin mRNA) in NBECs from 23 subjects without bronchogenic carcinoma compared to those from 11 subjects with bronchogenic carcinoma respectively (in parentheses) were: mGST (26.0, 6.11), GSTM3 (0.29, 0.09), combined GSTM1,2,4,5 (0.98, 0.60), GSTT1 (0.84, 0.76), GSTP1 (287, 110), GSHPx (140, 62.1), and GSHPxA (0.43, 0.34). Levels of GSTP1, GSTM3, and GSHPx were significantly (P < 0.05) lower in NBECs from subjects with bronchogenic carcinoma. Further, the gene expression index formed by multiplying the values for mGST x GSTM3 x GSHPx x GSHPxA x GSTP1 had a sensitivity (90%) and specificity (76%) for detecting NBECs from bronchogenic carcinoma subjects that was better than any individual gene. In cultured NBECs derived from eight individuals without bronchogenic carcinoma and incubated under identical conditions such that environmental effects were minimized, the mean level of expression and degree of interindividual variation for each gene evaluated was less than that observed in primary NBECs. Data from these studies support the hypotheses that (a) interindividual variation in risk for bronchogenic carcinoma results in part from interindividual variation in NBEC expression of antioxidant genes; (b) gene expression indices will better identify individuals at risk for bronchogenic carcinoma than individual gene expression values; and (c) both hereditary and environmental exposures contribute to the level of and interindividual variation in gene expression observed in primary NBECs. Many epidemiological studies have been designed to evaluate risk associated with polymorphisms or gene expression levels of putative susceptibility genes based on measurements in surrogate tissues, such as peripheral blood lymphocytes. Based on data presented here, it will be important to include the assessment of NBECs in future studies. Measurement of antioxidant gene expression in NBECs may identify the 5-10% of individuals at risk for bronchogenic carcinoma. Bronchoscopic sampling of NBECs from smokers and ex-smokers then will allow susceptible individuals to be entered into surveillance and/or chemoprevention studies.

Adolescent↗
PubMed1992

Gene-specific oligonucleotide probes for alpha, mu, pi, and microsomal rat glutathione S-transferases: analysis of liver transferase expression and its modulation by hepatic enzyme inducers and platinum anticancer drugs.

Glutathione S-transferases (GSTs) play an important role in the detoxification of diverse electrophilic chemicals, including anticancer drugs. Gene-specific oligonucleotide probes were developed to monitor the expression of individual GST mRNAs in livers of adult male rats treated with drugs and other chemical modulators of GST expression. Northern blot analysis of total liver RNA using probes specific for individual GSTs belonging to classes alpha (GSTs Ya1, Ya2, Yc), mu (GSTs Yb1, Yb2, Yb3), pi (GST Yp), and GSTms demonstrated the expression in liver of all but Yp mRNA. Kidney GST expression was at least as high as that in liver for GSTs Ya1, Yc, and Yp, while it was substantially lower but still detectable for GSTs Ya2, Yb2, and GSTms. Several of the liver GST class alpha mRNAs, in particular Ya2, were inducible by pretreatment of rats with phenobarbital or isosafrole. In contrast, dexamethasone preferentially induced Yb1, Yb2, and Ya2, while two other inducers of liver drug metabolism, isoniazid and clofibrate, were less effective with respect to GST induction. GSTms mRNA was induced to a small extent or not at all by the agents tested. Treatment of adult male rats with the anticancer drug cisplatin increased liver expression of GST Yc mRNA and suppressed Ya1 mRNA levels with little or no major effect on several other GST mRNAs. Western blot analysis of liver cytosols prepared from the cisplatin-treated rats revealed corresponding changes in GST Yc and Ya protein levels. Comparable changes in liver GST Ya1 and Yc expression were effected by the cisplatin analogue iproplatin but not by carboplatin or transplatin. This pattern of response to these platinum drugs is comparable to that seen with respect to platinum drug-induced gonadal toxicity and modulation of liver cytochrome P450 expression, suggesting a common mechanistic basis for these diverse effects of platinum anticancer drugs on hepatic enzymes of drug metabolism. Together, these studies demonstrate the utility of oligonucleotide probes for phenotyping liver tissue for the expression of GST enzymes that can contribute to anticancer drug metabolism and resistance. They also raise the possibility of drug-drug interactions involving cisplatin and alkylating agent anticancer drugs that can be metabolized in liver by alpha-class GSTs.

Animals↗
PubMed1990

Glutathione transferase activity in some flagellates and amoebae, and purification of the soluble glutathione transferases from Acanthamoeba.

Glutathione transferase (GST) enzymes are toxicologically important from many points of view. Nine protozoans were investigated here for their GST content. Six aerobic amoebae had very different specific GST activities, but an anaerobic amoeba and two anaerobic flagellates did not have any GST activity, suggesting that the peroxidase activity of GST is an evolutionarily important property for aerobic organisms. The soluble GST isoenzymes of Acanthamoeba culbertsoni and A. polyphaga were purified and partially characterized. The same two cationic and one anionic GST isoenzyme were found in both Acanthamoeba ssp., while A. culbertsoni had one more cationic isoenzyme. It is concluded that GST in aerobic amoebae can play an important role in detoxication.

Acanthamoeba↗
PubMed1986

Mechanism-based fragmentation of coenzyme A transferase. Comparison of alpha 2-macroglobulin and coenzyme A transferase thiol ester reactions.

The plasma proteins, alpha 2-macroglobulin and complement components 3 and 4, contain an internal thiol ester involving a glutamyl and cysteinyl residue. The thiol ester is susceptible to cyclization at greater than 37 degrees C and forms an unstable 5-oxyproline intermediate. The latter can be hydrolyzed to produce two peptide fragments. We propose that enzymes having activated glutamyl residues as part of their catalytic mechanisms may undergo an analogous cyclization and peptidyl cleavage. As a model, we have investigated pig heart succinyl-CoA:3-keto acid transferase. When the CoA-enzyme thiolester intermediate is heated at pH 7.4 and 70 degrees C for 1 h, approximately 60% of the Mr = 60,000 subunits are cleaved to give Mr = 40,000 and 20,000 fragments. We have shown that formation of the enzyme thiolester is an obligate precursor for the protein fragmentation. However, the reaction was incomplete with a maximum of approximately 65% cleavage at times greater than 60 min. These results suggest that there is a competing, deactivation reaction; namely, the thiol ester and oxyproline intermediates are hydrolyzed to regenerate the active site glutamic acid. Although the maximum rate of cleavage is at 70 degrees C, approximately 15% autolysis also occurs at 37 degrees C. The Mr = 40,000 fragment had the same amino terminal sequence as the Mr = 60,000 subunit, (Trp-Lys-Phe-Tyr-Thr-Asp-Ala-Val-Glu-Ala-). No amino terminal could be detected for the Mr = 20,000 fragment, even after digesting the fragment with pyroglutaminase. Peptide maps of the fragments and the uncleaved subunit indicate that the fragments are generated in parallel. The size of the fragments puts the active site about two-thirds of the way from the amino terminal of the protein.

Acyltransferases↗