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PubMed1997

Evidence that human class Theta glutathione S-transferase T1-1 can catalyse the activation of dichloromethane, a liver and lung carcinogen in the mouse. Comparison of the tissue distribution of GST T1-1 with that of classes Alpha, Mu and Pi GST in human.

The cDNA encoding human glutathione S-transferase (GST) T1 has been expressed as two recombinant forms in Escherichia coli that could be purified by affinity chromatography on either IgG-Sepharose or nickel-agarose; one form of the transferase was synthesized from the pALP 1 expression vector as a Staphylococcus aureus protein A fusion, whereas the other form was synthesized from the pET-20b expression vector as a C-terminal polyhistidine-tagged recombinant. The yields of the two purified recombinant proteins from E. coli cultures were approx. 15 mg/l for the protein A fusion and 25 mg/l for the C-terminal polyhistidine-tagged GST T1-1. The purified recombinant proteins were catalytically active, although the protein A fusion was typically only 5-30% as active as the histidine-tagged GST. Both recombinant forms could catalyse the conjugation of glutathione with the model substrates 1,2-epoxy-3-(4'-nitrophenoxy)propane,4-nitrobenzyl chloride and 4-nitrophenethyl bromide but were inactive towards 1-chloro-2,4-dinitrobenzene, ethacrynic acid and 1-menaphthyl sulphate. Recombinant human GST T1-1 was found to exhibit glutathione peroxidase activity and could catalyse the reduction of cumene hydroperoxide. In addition, recombinant human GST T1-1 was found to conjugate glutathione with dichloromethane, a pulmonary and hepatic carcinogen in the mouse. Immunoblotting with antibodies raised against different transferase isoenzymes showed that GST T1-1 is expressed in a large number of human organs in a tissue-specific fashion that differs from the pattern of expression of classes Alpha, Mu and Pi GST. Most significantly, GST T1-1 was found in only low levels in human pulmonary soluble extract of cells, suggesting that in man the lung has little capacity to activate the volatile dichloromethane.

Amino Acid Sequence↗
PubMed1998

Glutathione transferase zeta catalyses the oxygenation of the carcinogen dichloroacetic acid to glyoxylic acid.

Dichloroacetic acid (DCA), a common drinking-water contaminant, is hepatocarcinogenic in rats and mice, and is a therapeutic agent used clinically in the management of lactic acidosis. DCA is biotransformed to glyoxylic acid by glutathione-dependent cytosolic enzymes in vitro and is metabolized to glyoxylic acid in vivo. The enzymes that catalyse the oxygenation of DCA to glyoxylic acid have not, however, been identified or characterized. In the present investigation, an enzyme that catalyses the glutathione-dependent oxygenation of DCA was purified to homogeneity (587-fold) from rat liver cytosol. SDS/PAGE and HPLC gel-filtration chromatography showed that the purified enzyme had a molecular mass of 27-28 kDa. Sequence analysis showed that the N-terminus of the purified protein was blocked. An internal sequence of 30 amino acid residues was obtained that matched the recently discovered human glutathione transferase Zeta well [Board, Baker, Chelvanayagam and Jermiin (1997) Biochem. J. 328, 929-935]. Western-blot analysis showed that the purified rat-liver enzyme cross-reacted with rabbit antiserum raised against recombinant human glutathione transferase Zeta. The apparent Km and Vmax values of the purified enzyme with DCA as the variable substrate were 71.4 microM and 1334 nmol/min per mg of protein, respectively; the Km for glutathione was 59 microM. Both the purified rat-liver enzyme and the recombinant human enzyme showed high activity with DCA as the substrate. These results demonstrate that the glutathione-dependent oxygenation of DCA to glyoxylic acid is catalysed by a Zeta-class glutathione transferase.

Amino Acid Sequence↗
PubMed1998

Thioltransferase activity of bovine lens glutathione S-transferase.

A Mu-class glutathione S-transferase purified to electrophoretic homogeneity from bovine lens displayed thioltransferase activity, catalysing the transthiolation reaction between GSH and hydroxyethyldisulphide. The thiol-transfer reaction is composed of two steps, the formation of GSSG occurring through the generation of an intermediate mixed disulphide between GSH and the target disulphide. Unlike glutaredoxin, which is only able to catalyse the second step of the transthiolation process, glutathioneS-transferase catalyses both steps of the reaction. Data are presented showing that bovine lens glutathione S-transferase and rat liver glutaredoxin, which was used as a thioltransferase enzyme model, can operate in synergy to catalyse the GSH-dependent reduction of hydroxyethyldisulphide.

Animals↗
PubMed1999

Distribution of microsomal glutathione transferase 1 in mammalian tissues. A predominant alternate first exon in human tissues.

An extensive Northern blot analysis of microsomal glutathione transferase 1 in human and rat tissues was performed. When normalized against the glyceraldehyde-3-phosphate dehydrogenase or actin expression it was evident that the predominant expression occurs in liver and pancreas. An ontogenetic, as well as a functional, basis for the high levels in these two organs is possible. The relative expression levels in man ranged from: liver and pancreas (100%), to kidney, prostate, colon (30-40%), heart, brain, lung, testis, ovary, small intestine (10-20%), placenta, skeletal muscle, spleen, thymus and peripheral blood leucocytes (1-10%). Liver-enriched expression was detected in human fetal tissues with lung and kidney displaying lower levels (10-20%). No transcripts could be detected in fetal brain or heart. When comparing the expression levels between rat and man it is apparent that human extrahepatic mRNA levels are much higher relative to liver. Rat microsomal glutathione transferase mRNA expression ranges from 0.2 to 10% that of liver, with adrenal, uterus, ovary and stomach displaying the highest levels of the organs tested. Based on these observations, and the fact that the enzyme is encoded by a highly conserved single-copy gene, it is suggested that microsomal glutathione transferase 1 performs essential functions vital to most mammalian cell types. We suggest that protection against oxidative stress constitutes one such function. Human expressed sequence tag (EST) characterization yielded four alternate mRNA transcripts with different 5'-ends (four alternate noncoding exons 1). The predominant exon (based on the observed EST frequency) revealed a tissue distribution similar to that obtained using the reading frame as probe. Thus, it appears that one exon preferentially gives rise to mature mRNA in the human tissues examined. This exon is different from the one reported in the original cDNA characterized.

Animals↗
PubMed2001

Catalytic function of Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (GST-2) in conjugation of lipid peroxidation end products.

Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (earlier designated as GST-2) is related to sigma class GSTs and was previously described as an indirect flight muscle-associated protein with no known catalytic properties. We now report that DmGSTS1-1 isolated from Drosophila or expressed in Escherichia coli is essentially inactive toward the commonly used synthetic substrate 1-chloro-2,4-dinitrobenzene (CDNB), but has relatively high glutathione-conjugating activity for 4-hydroxynonenal (4-HNE), an electrophilic aldehyde derived from lipid peroxidation. 4-HNE is thought to have signaling functions and, at higher concentrations, has been shown to be cytotoxic and involved in the etiology of various degenerative diseases. Drosophila strains carrying P-element insertions in the GstS1 gene have a reduced capacity for glutathione conjugation of 4-HNE. In flies with both, one, or none of the GstS1 alleles disrupted by P-element insertion, there is a linear correlation between DmGSTS1-1 protein content and 4-HNE-conjugating activity. This correlation indicates that in adult Drosophila 70 +/- 6% of the capacity to conjugate 4-HNE is attributable to DmGSTS1-1. The high abundance of DmGSTS1-1 (approximately 2% of the soluble protein in adult flies) and its previously reported localization in tissues that are either highly aerobic (indirect flight muscle) or especially sensitive to oxidative damage (neuronal tissue) suggest that the enzyme may have a protective role against deleterious effects of oxidative stress. Such function in insects would be analogous to that carried out in mammals by specialized alpha class glutathione S-transferases (e.g. GSTA4-4). The independent emergence of 4-HNE-conjugating activity in more than one branch of the glutathione S-transferase superfamily suggests that 4-HNE catabolism may be essential for aerobic life.

Aldehydes↗
PubMed2000

Farnesyl transferase inhibitors: current developments and future perspectives.

Ras oncogenes play an important role in carcinogenesis and are frequently found in various human tumour types. Cellular activity of Ras oncoprotein, regulated through the enzyme farnesyl transferase, is crucial in the process of ras -dependent carcinogenesis, and therefore, specific inhibition of this enzyme is an attractive goal in anticancer treatment. Specific inhibitors of farnesyl transferase have been developed in recent years, many of them showing in vitro and in vivo growth inhibitory or cytostatic activity. Recently, results of the first clinical studies with various farnesyl transferase inhibitors have been presented. In the design of phase I and II studies, either single-agent or combination studies, new endpoints have to be defined in order to properly assess feasibility, antitumour activity and clinical valuability.

Alkyl and Aryl Transferases↗
PubMed2005

Telomerase can act as a template- and RNA-independent terminal transferase.

Telomerase is a special reverse transcriptase that extends one strand of the telomere repeat by using a template embedded in an RNA subunit. Like other polymerases, telomerase is believed to use a pair of divalent metal ions (coordinated by a triad of aspartic acid residues) for catalyzing nucleotide addition. Here we show that, in the presence of manganese, both yeast and human telomerase can switch to a template- and RNA-independent mode of DNA synthesis, acting in effect as a terminal transferase. Even as a terminal transferase, yeast telomerase retains a species-dependent preference for GT-rich, telomere-like DNA on the 5' end of the substrate. The terminal transferase activity of telomerase may account for some of the hitherto unexplained effects of telomerase overexpression on cell physiology.

Base Sequence↗
PubMed1977

Translation in vitro of rat liver messenger RNA coding for ligandin (glutathione S-transferase B).

Poly(A)-containing rat liver mRNA isolated from animals injected with phenobarbital and uninjected controls was translated efficiently in a wheat-germ system. The synthesis of ligandin (glutathione S-transferase B; glutathione transferase; RX-gluathione R-transferase, EC 2.5.1.18) was detected by immunoprecipitation with a highly purified monospecific ligandin antibody and analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The extent of incorporation of [35S]methionine into ligandin in the translation system was similar for poly(A)-containing messages from un-infected animals and those treated with phenobarbital.

Animals↗
PubMed1985

Purification, induction, and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis.

A polypeptide of Mr 26,000 and pI 6.7 that was markedly increased in rat livers bearing hyperplastic nodules (HNs) induced by chemical carcinogens was identified immunochemically as the subunit of neutral glutathione (GSH) transferase (GSHTase; RX:glutathione R-transferase, EC 2.5.1.18; also called GSH S-transferase) purified from placenta (GSHTase-P) and was demonstrated immunohistochemically to be localized in preneoplastic foci and HNs. In the present study, GSHTase-P has been purified from the HN-bearing liver, and the distribution and inducibility have been examined quantitatively using anti-GSHTase-P antibody. Elevation of GSHTase-P in the HN-bearing livers was also confirmed by in vitro translation of mRNAs isolated from the HN-bearing livers. The purified GSHTase-P was homogeneous in size but had two charge isomers on two-dimensional gel electrophoresis. In normal tissues, including liver, placenta, and fetal liver, the protein content of GSHTase-P was generally low but was significantly high in kidney and pancreas. In contrast, the amount of GSHTase-P in HN-bearing livers (primary hepatomas) and transplantable Morris hepatoma 5123D were several 10-fold higher than that in normal liver but were undetectably low in transplantable Yoshida ascites hepatoma AH 130. Different from ordinary drug-metabolizing enzymes, GSHTase-P was uninducible by administration of drugs and carcinogens prior to appearance of the preneoplastic foci and HNs. In addition, species specificity of GSHTase-P was low as it was crossreactive among rat, hamster, and human.

Amino Acids↗
PubMed1986

Mr 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase.

Mice of the inbred strain 129/J bred at this Institute (WEHI 129/J) are relatively resistant to chronic infection with the parasitic helminth Schistosoma japonicum. In contrast to more permissive mouse strains such as BALB/c, the WEHI 129/J mice are high responders to a Mr 26,000 adult worm antigen designated Sj26. Cloned cDNAs corresponding to Sj26 have been identified in a S. japonicum phage lambda gt11 amp3 expression library, and their nucleotide sequences have been deduced. The predicted amino acid sequence of the antigen specified by these cDNAs shows striking homology with class mu isozymes of mammalian glutathione S-transferases (RX:glutathione R-transferase, EC 2.5.1.18). Extracts of adult worms contain glutathione S-transferase activity, and affinity chromatography of enzyme activity on glutathione columns leads to the purification of a Mr 26,000 molecule that comigrates with Sj26. Although vaccination studies in mice with a beta-galactosidase-Sj26 fusion protein from Escherichia coli are encouraging, more immunogenic preparations of the antigen are likely to be required to establish the utility of Sj26 as a model vaccine.

Amino Acid Sequence↗
PubMed1986

Structural analysis of a rat liver glutathione S-transferase Ya gene.

We have isolated and characterized a complete structural gene encoding a rat liver glutathione S-transferase (glutathione transferase; EC 2.5.1.18) Ya subunit. The gene spans approximately 11 kilobases and is comprised of seven exons separated by six introns. A sequence similar to the Goldberg-Hogness promoter ("TATA" box), TATTA, is located 32 base pairs upstream from the transcription initiation site. Exons 2 and 4 of the glutathione S-transferase gene encode amino acid sequences of the Ya subunit that are highly conserved in the Yc subunit, whereas exons 3 and 5 encode amino acids that are divergent in the Yc subunit. These data suggest that exons 2 and 4 may encode domains of the Ya subunits that have similar structural or functional properties to the corresponding domains in the Yc subunit (e.g., glutathione binding site), whereas exons 3 and 5 may encode domains of the Ya subunit that have unique structural or functional properties to the corresponding domains in the Yc subunit (e.g., substrate binding site).

Animals↗
PubMed1991

Cloning, expression, and characterization of a class-mu glutathione transferase from human muscle, the product of the GST4 locus.

A class-mu glutathione transferase cDNA clone, GTHMUS, was isolated from human myoblasts and its sequence was determined. The sequence predicts a protein of molecular weight 25,599 whose 24 amino-terminal residues are identical to those of the class-mu isoenzyme expressed from the GST4 locus. The GTHMUS cDNA shares 93.7% nucleotide sequence identity with a human liver cDNA clone, GTH411, that is encoded at the GST1 locus. Comparison of the liver and muscle cDNA sequences shows two regions of remarkable sequence conservation: a 140-nucleotide region in the 5' coding portion of the molecule that has a single silent nucleotide substitution, and a 550-nucleotide region, including the entire 3' noncoding region, that has only three nucleotide substitutions or deletions. This sequence conservation suggests that gene conversion has occurred between the human GST1 and GST4 glutathione transferase gene loci. The human muscle and liver glutathione transferase clones GTHMUS and GTH411 have been expressed in Escherichia coli. The kinetic mechanism of the muscle enzyme was examined in product inhibition studies. The inhibition patterns are best modeled by a steady-state ordered bi-bi reaction mechanism. Glutathione is the first substrate bound and chloride ion is the first product released. Chloride ion inhibits the muscle enzyme.

Amino Acid Sequence↗
PubMed1993

Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes.

Dihalomethanes can produce liver tumors in mice but not in rats, and concern exists about the risk of these compounds to humans. Glutathione (GSH) conjugation of dihalomethanes has been considered to be a critical event in the bioactivation process, and risk assessment is based upon this premise; however, there is little experimental support for this view or information about the basis of genotoxicity. A plasmid vector containing rat GSH S-transferase 5-5 was transfected into the Salmonella typhimurium tester strain TA1535, which then produced active enzyme. The transfected bacteria produced base-pair revertants in the presence of ethylene dihalides or dihalomethanes, in the order CH2Br2 > CH2BrCl > CH2Cl2. However, revertants were not seen when cells were exposed to GSH, CH2Br2, and an amount of purified GSH S-transferase 5-5 (20-fold excess in amount of that expressed within the cells). HCHO, which is an end product of the reaction of GSH with dihalomethanes, also did not produce mutations. S-(1-Acetoxymethyl)GSH was prepared as an analog of the putative S-(1-halomethyl)GSH reactive intermediates. This analog did not produce revertants, consistent with the view that activation of dihalomethanes must occur within the bacteria to cause genetic damage, presenting a model to be considered in studies with mammalian cells. S-(1-Acetoxymethyl)GSH reacted with 2'-deoxyguanosine to yield a major adduct, identified as S-[1-(N2-deoxyguanosinyl)methyl]GSH. Demonstration of the activation of dihalomethanes by this mammalian GSH S-transferase theta class enzyme should be of use in evaluating the risk of these chemicals, particularly in light of reports of the polymorphic expression of a similar activity in humans.

Animals↗
PubMed1994

Anti-peptidyl transferase leader peptides of attenuation-regulated chloramphenicol-resistance genes.

The chloramphenicol (Cm)-inducible cmlA gene of Tn1696 specifies nonenzymatic resistance to Cm and is regulated by attenuation. The first eight codons of the leader specify a peptide that inhibits peptidyl transferase in vitro. Functionally similar, but less inhibitory, peptides are encoded by the leaders of Cm-inducible cat genes. However, the cat and cmlA coding sequences are unrelated and specify proteins of unrelated function. The inhibition of peptidyl transferase by the leader peptides is additive with that of Cm. Erythromycin competes with the inhibitory action of the peptides, and erythromycin and the peptides footprint to overlapping sites at the peptidyl transferase center of 23S rRNA. It is proposed that translation of the cmlA and cat leaders transiently pauses upon synthesis of the inhibitor peptides. The predicted site of pausing is identical to the leader site where long-term occupancy by a ribosome (ribosome stalling) will activate downstream gene expression. We therefore propose the inducer, Cm, converts a peptide-paused ribosome to the stalled state. We discuss the idea that cooperativity between leader peptide and inducer is necessary for ribosome stalling and may link the activation of a specific drug-resistance gene with a particular antibiotic.

Amino Acid Sequence↗
PubMed1994

Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases.

Radiation and chemical reactions that give rise to free radicals cause the formation of highly cytotoxic base propenals, degradation products of DNA. Human glutathione transferases (GSTs; RX:glutathione R-transferase, EC 2.5.1.18) of classes Alpha, Mu, and Pi were shown to promote the conjugation of glutathione with base propenals and related alkenes. GST P1-1 was particularly active in catalyzing the reactions with the propenal derivatives, and adenine propenal was the substrate giving the highest activity. The catalytic efficiency of GST P1-1 with adenine propenal (kcat/Km = 7.7 x 10(5) M-1.s-1) is the highest so far reported with any substrate for this enzyme. In general, GST A1-1 and GST M1-1, in contrast to GST P1-1, were more active with 4-hydroxyalkenals (products of lipid peroxidation) than with base propenals. The adduct resulting from the Michael addition of glutathione to the alkene function of one of the base propenals (adenine propenal) was identified by mass spectrometry. At the cellular level, GST P1-1 was shown to provide protection against alpha, beta-unsaturated aldehydes. GST P1-1 added to the culture medium of HeLa cells augmented the protective effect of glutathione against the toxicity of adenine propenal and thymine propenal. No protective effect of the enzyme was observed in the presence of the competitive inhibitor S-hexylglutathione. GST P1-1 introduced into Hep G2 cells by electroporation was similarly found to increase their resistance to acrolein. The results show that glutathione transferases may play an important role in cellular detoxication of electrophilic alpha, beta-unsaturated carbonyl compounds produced by radical reactions, lipid peroxidation, ionizing radiation, and drug metabolism.

Acrolein↗
PubMed1997

Identification and characterization of a novel microsomal enzyme with glutathione-dependent transferase and peroxidase activities.

5-Lipoxygenase activating protein (FLAP), leukotriene-C4 (LTC4) synthase, and microsomal glutathione S-transferase II (microsomal GST-II) are all members of a common gene family that may also include microsomal GST-I. The present work describes the identification and characterization of a novel member of this family termed microsomal glutathione S-transferase III (microsomal GST-III). The open reading frame encodes a 16.5-kDa protein with a calculated pI of 10.2. Microsomal GST-III has 36, 27, 22, and 20% amino acid identity to microsomal GST-II, LTC4 synthase, microsomal GST-I, and FLAP, respectively. Microsomal GST-III also has a similar hydrophobicity pattern to FLAP, LTC4 synthase, and microsomal GST-I. Fluorescent in situ hybridization mapped microsomal GST-III to chromosomal localization 1q23. Like microsomal GST-II, microsomal GST-III has a wide tissue distribution (at the mRNA level) and is predominantly expressed in human heart, skeletal muscle, and adrenal cortex, and it is also found in brain, placenta, liver, and kidney tissues. Expression of microsomal GST-III mRNA was also detected in several glandular tissues such as pancreas, thyroid, testis, and ovary. In contrast, microsomal GST-III mRNA expression was very low (if any) in lung, thymus, and peripheral blood leukocytes. Microsomal GST-III protein was expressed in a baculovirus insect cell system, and microsomes from Sf9 cells containing either microsomal GST-II or microsomal GST-III were both found to possess glutathione-dependent peroxidase activity as shown by their ability to reduce 5-HPETE to 5-HETE in the presence of reduced glutathione. The apparent Km of 5-HPETE was determined to be approximately 7 microM for microsomal GST-II and 21 microM for microsomal GST-III. Microsomal GST-III was also found to catalyze the production of LTC4 from LTA4 and reduced glutathione. Based on these catalytic activities it is proposed that this novel membrane protein is a member of the microsomal glutathione S-transferase super family, which also includes microsomal GST-I, LTC4 synthase, FLAP, and microsomal GST-II.

Amino Acid Sequence↗
PubMed1998

Ribosomal protein L27 participates in both 50 S subunit assembly and the peptidyl transferase reaction.

Protein L27 has been implicated as a constituent of the peptidyl transferase center of the Escherichia coli 50 S ribosomal subunit by a variety of experimental observations. To define better the functional role of this protein, we constructed a strain in which the rpmA gene, which encodes L27, was replaced by a kanamycin resistance marker. The deletion mutant grows five to six times slower than the wild-type parent and is both cold- and temperature-sensitive. This phenotype is reversed when L27 is expressed from a plasmid-borne copy of the rpmA gene. Analysis of ribosomes from the L27-lacking strain revealed deficiencies in both the assembly and activity of the 50 S ribosomal subunits. Although functional 50 S subunits are formed in the mutant, an assembly "bottleneck" was evidenced by the accumulation of a prominent 40 S precursor to the 50 S subunit which was deficient in proteins L16, L20, and L21, as well as L27. In addition, the peptidyl transferase activity of 70 S ribosomes containing mutant 50 S subunits was determined to be three to four times lower than for wild-type ribosomes. Ribosomes lacking L27 were found to be impaired in the enzymatic binding of Phe-tRNAPhe to the A site, although the interaction of N-acetyl-Phe-tRNAPhe with the P site was largely unperturbed. We therefore infer that L27 contributes to peptide bond formation by facilitating the proper placement of the acceptor end of the A-site tRNA at the peptidyl transferase center.

Binding Sites↗
PubMed2000

Identification, characterization, and crystal structure of the Omega class glutathione transferases.

A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species and Caenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-A resolution and has a characteristic GST fold (Protein Data Bank entry code ). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.

Amino Acid Sequence↗