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At least 685 records · Page 38Linked to original sources
PubMed1991

Activities of glutathione-S-transferase and ethoxycoumarin-O-deethylase in tissues of camels, sheep, goats and rats.

1. The activities of the drug metabolizing enzymes ethoxycoumarin-O-deethylase, glutathione-S-transferase, and protein concentrations were measured in vitro in the liver, kidney and duodenal mucosa of camels, sheep, goats and rats. 2. Enzyme activities were generally higher in the liver than in the kidney and duodenal mucosa in the four species studied. 3. The activities of ethoxycoumarin-O-deethylase and glutathione-S-transferase in liver of male kids were about one third and half of that in adult male goats, respectively. In the kidney and duodenal mucosa of male kids, the activity of glutathione-S-transferase was about 70% and 53% of that in the mature male goat, respectively. In the latter tissues, however, there was no detectable activity of ethoxycoumarin-O-deethylase. 4. In general, goats and sheep had similar activities of the two enzymes which were significantly higher than those found in camels and rats. 5. Some sex-related differences were noted in the activity of the two enzymes studied. Female sheep had significantly higher hepatic glutathione-S-transferase than the male: while the enzyme activity in the kidney and duodenal mucosa of male goats was significantly higher than in females. Male rats had higher hepatic ethoxycoumarin-O-deethylase activity than females.

7-Alkoxycoumarin O-Dealkylase↗
PubMed2004

Expression of selenocysteine-containing glutathione S-transferase in Escherichia coli.

Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST), having glutathione peroxidase (GPX) activity. Here, we addressed this question by production of such protein. In order to obtain a recombinant seleno-GST produced in Escherichia coli, we introduced a variant bacterial-type selenocysteine insertion sequence (SECIS) element which afforded substitution with selenocysteine for the catalytic Tyr residue in the active site of GST from Schistosoma japonica. Utilizing coexpression with the bacterial selA, selB, and selC genes (encoding selenocysteine synthase, SelB, and tRNA(Sec), respectively) the yield of recombinant seleno-GST was about 2.9 mg/L bacterial culture, concomitant with formation of approximately 85% truncation product as a result of termination of translation at the selenocysteine-encoding UGA codon. The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione-binding capacity (Km = 53 microM for glutathione as compared to 63 microM for the wild-type enzyme) nor the GST activity (kcat = 14.3 s(-1) vs. 16.6 s(-1)), provided that the catalytic Tyr residue was intact. When this residue was changed to selenocysteine, however, the resulting seleno-GST lost the GST activity. It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide). These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E. coli at sufficient amounts for purification. We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione-binding capacity.

Amino Acid Sequence↗
PubMed2006

Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities.

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.

Animals↗
PubMed1992

Drosophila glutathione S-transferases have sequence homology to the stringent starvation protein of Escherichia coli.

The Drosophila glutathione S-transferase D genes encode a family of isozymes. We have determined the amino acid sequence of a new member of this family by nucleotide sequence analysis of a genomic DNA clone. The open reading frame of this intronless gene should encode an isozyme subunit of 211 amino acids. This sequence has significant homology to the E. coli stringent starvation protein, SSP, which is also a protein of two identical 211 amino acid subunits. The two proteins have very similar overall amino acid composition as well. It is possible that SSP may be a glutathione S-transferase(s) in E. coli or is evolutionarily related to glutathione S-transferases. Because SSP is known to be tightly associated with the RNA polymerase holoenzyme during purification, it is conceivable that Drosophila glutathione S-transferase(s) may potentially interact with the transcription machinery in a fashion similar to SSP's interaction with E. coli RNA polymerase holoenzyme.

Amino Acid Sequence↗
PubMed1985

Differences in the occurrence of glutathione transferase isoenzymes in rat lung and liver.

Cytosolic GSH transferases have been purified from rat lung by affinity chromatography followed by chromatofocusing. On the criteria of order of elution, substrate specificity, apparent subunit Mr, sensitivity to inhibitors, and reaction with antibodies, transferase subunits equivalent to subunits 2, 3, and 4, in the binary combinations occurring in liver, were identified. However, subunit 1 (and therefore transferases 1-1 and 1-2) was not detected. The most conspicuous difference is the presence in lung of a new form, eluting at pH 8.7, which is not detected in rat liver. This isoenzyme (transferase "pH 8.7") is characterized by its low apparent subunit Mr and high efficiency in the conjugation of glutathione with anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, considered the ultimate carcinogen of benzo(a)-pyrene.

Animals↗
PubMed1989

Amino acid substitutions in the human glutathione S-transferases confer different specificities in the prostaglandin endoperoxide conversion pathway.

The human glutathione S-transferases 1-1 and 2-2, which differ from each other by 11 amino acids, have different catalytic activities against cumene hydroperoxide and t-butyl hydroperoxide. Using prostaglandin H2 as the peroxide substrate, we found that GSH S-transferase 1-1 catalyzed the transformation of prostaglandin H2 to prostaglandin F2 alpha and E2 at a 4:1 ratio whereas GSH S-transferase 2-2 produced primarily prostaglandin D2 and F2 alpha at a 4:1 ratio. Our results indicate that GSH S-transferases catalyze the reduction and isomerization of prostaglandin H2 endoperoxide in vitro. We suggest that the amino acid substitutions between these two isozymes may be responsible for the difference in catalytic specificities. We propose that these isozymes are important reagents for the biosynthesis of various prostaglandins.

Dinoprost↗
PubMed1998

Mechanistic imperative for the evolution of a metalloglutathione transferase of the vicinal oxygen chelate superfamily.

A number of glutathione (GSH) transferases are now known in prokaryotes and eukaryotes. The enzymes appear to be primarily involved in the metabolism of foreign compounds. At least six distinct classes of soluble GSH transferases have been identified in eukaryotes and named alpha, mu, pi, sigma, theta and kappa. Sequences and the known three-dimensional structures of the soluble enzymes suggest that these proteins share a common ancestry, though the precise details of their evolution remain obscure. A second distinct family of GSH transferases are the microsomal or membrane-bound enzymes that include leukotriene C4 synthase. A third family is represented by a bacterial GSH transferase (FosA) responsible for conferring resistance to the antibiotic fosfomycin, reported some years ago by Suarez and co-workers (Arca et al., Antimicrob. Agents Chemother. 34 (1990) 1552-1556). The enzyme is quite specific for fosfomycin, which contains a very stable epoxide moiety. Evidence is presented that FosA is a metalloprotein related to iron- and manganese-dependent dioxygenases and to glyoxalase I. These enzymes are members of a previously unrecognized group of enzymes; the vicinal oxygen chelate superfamily. The mechanistic imperative driving the evolution of FosA and its relatives, which are enzymes catalyzing quite diverse chemical reactions, is proposed to be the electrophilic assistance provided by the metal through chelation of a substrate or intermediate.

Animals↗
PubMed2000

Colocalization of leukotriene C synthase and microsomal glutathione S-transferase elucidated by indirect immunofluorescence analysis.

We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S-transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well-known sources of leukotriene C synthase, both expressed microsomal glutathione S-transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S-transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S-transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein-protein interaction may contribute to the regulation of LTC4 production.

Animals↗
PubMed1987

Müllerian aplasia associated with maternal deficiency of galactose-1-phosphate uridyl transferase.

The activity and electrophoretic pattern of galactose-1-phosphate uridyl transferase (transferase), a key enzyme in galactose metabolism, were analyzed in four patients with Müllerian aplasia (Rokitansky-Küster-Hauser syndrome) and their mothers. Mothers of two of the patients had genetic variations of their transferase enzymes with activities below the normal range. Affected daughters from these two mothers also had genetic variations of the transferase enzyme. In one of the patients whose Müllerian aplasia had been diagnosed 15 years previously, premature ovarian failure developed. These case reports suggest a possible association between errors of galactose metabolism, Müllerian aplasia, and premature menopause--an association that is supported by a rodent model in which female offspring of mothers fed a high-galactose diet were born with reduced oocyte numbers and delayed vaginal opening.

Adult↗
PubMed1986

Anion-exchange high-performance liquid chromatography of glutathione S-transferases. Separation of the minor isoenzymes of human erythrocyte, heart and lung.

Apparently homogeneous preparation of the acidic glutathione S-transferases were subjected to high-performance liquid chromatography over a SynChropak AX-300 anion-exchange column. Results of high-performance liquid chromatography analysis indicated that in each of these tissues the acidic glutathione S-transferases have one major and two minor isoenzymes. All the isoenzymes obtained by high-performance liquid chromatography are dimers of subunits of molecular weight (Mr) 22,500. The major isoenzymes of all the tissues, representing more than 80% of total glutathione S-transferase activity, have similar retention times and comparable kinetic properties. High-performance liquid chromatography profiles of the lung and heart isoenzymes are similar to each other but are significantly different from that of the erythrocytes. These studies provide evidence of microheterogeneity in the acidic isoenzymes of heart, lung and erythrocyte glutathione S-transferases and provide a rapid and efficient method for separation of minor isoenzymes in more than 95% yield.

Blood Protein Electrophoresis↗
PubMed1995

Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1.

Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.

Arginine↗
PubMed1993

Enhanced glutathione S-transferase activity and glutathione content in human bladder cancer. Followup study: influence of smoking.

Glutathione content and glutathione S-transferase activity have been studied in human bladder specimens obtained from controls and from patients with superficial transitional cell carcinoma (tumor samples and peri-tumor normal tissues from the same patient). After combining an earlier study from our laboratory with the additional material presented (9 healthy controls and 25 transitional cell carcinoma patients), it can be observed that glutathione S-transferase activity was significantly greater in tumor than in peri-tumor normal tissue (34 patients, p < 1 x 10(-7)) or in normal mucosa (17 controls, p < 1 x 10(-3)). Glutathione content was significantly greater in tumor than in peri-tumor normal tissue (p < 5 x 10(-3)) or in normal mucosa (p < 2 x 10(-2)), with this increase being evident only in smokers. When comparing normal mucosa and peri-tumor samples no significant differences were found either for glutathione S-transferase activity or for glutathione content. Results demonstrate the relationship between the glutathione S-transferase/glutathione system and development of transitional cell carcinoma, as well as its role in cellular resistance to chemotherapy.

Aged↗
PubMed1998

Differential induction of glutathione S-transferase subunits by spironolactone in rat liver, jejunum and colon.

The effect of spironolactone pretreatment on glutathione S-transferase activity and on the relative content of the principal subunits (Ya, Yc, Yb1, Yb2 and Yp or 1, 2, 3, 4 and 7 respectively) was studied in rat liver, jejunum and colon. Male Wistar rats were injected with spironolactone i.p. at daily doses of 50, 100 and 200 micromol/kg body wt for 3 consecutive days. Glutathione S-transferase activities were assayed using 1-chloro-2,4-dinitrobenzene as substrate. Changes in subunit composition were evaluated by Western blot analysis in rats treated with the highest dose of spironolactone. The results demonstrated a dose-dependent increase in enzyme activity in liver, while in jejunum the three tested doses exhibited the same magnitude of induction. No significant difference in glutathione S-transferase activity was observed between control and treated rats for the colon. Immunoblot analysis revealed more Ya and Yp protein in liver (140 and 118% increase respectively) and jejunum (45 and 145% increase respectively) from treated rats. While Ya and Yp relative contents were similar in jejunum, the latter subunit slightly contributed to total GST in liver, even in SL-treated animals. The inducer produced no change in subunit composition in colon. In conclusion, spironolactone was able to increase glutathione S-transferase activity mainly by induction of Ya subunit in liver and Yp subunit in jejunal mucosa, without affecting colonic enzyme.

Animals↗
PubMed2005

Microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) is an abundant membrane-bound glutathione transferase and peroxidase constituting 3% of the endoplasmic reticulum protein in rat liver (and 5% of the outer mitochondrial membrane). The enzyme is most well studied in mammals and belongs to a large and widely distributed superfamily. Cellular and organelle protection versus oxidative stress has been demonstrated. The enzyme displays activity to a multitude of reactive substrates ranging from products of lipid peroxidation to cytostatic drugs. The methods developed for the study of MGST1 by necessity differs from that of cytosolic glutathione transferases, because detergents or lipids are included. Here, purification, assay, and preparation procedures that maintain the enzyme in its native functional state during isolation and characterization are described. Microsomal glutathione transferase 1 is activated by sulfhydryl reagents (and proteolysis), and procedures for activation and study of the activated enzyme are described. In new developments, the enzyme is studied by pre-steady state methods, as well as mass spectrometry involving direct observation of the native enzyme.

Animals↗
PubMed1998

Alpha-glutathione transferases in HCV-related chronic hepatitis: a new predictive index of response to interferon therapy?

BACKGROUND/AIM: The aim of this study was to evaluate if plasma levels of alpha-glutathione-S-transferases (determined in basal conditions and monthly for 1 year during and 1 year after interferon therapy) could characterize patients who show only a primary response. METHODS: We studied 48 patients with biopsy-proven, hepatitis C virus ribonucleic acid positive chronic hepatitis treated with interferon: 18 were "Sustained Responders", 12 "Relapsers" and 18 "Non-Responders". RESULTS: Relapsers showed higher basal levels of alpha-glutathione-S-transferases, which remained higher than normal even when alanine aminotransferases normalized. No correlation was documented between alpha-glutathione-S-transferase levels and all other parameters examined (alanine aminotransferases, gamma-glutamyl-transpeptidase, viremia, and histological activity index). CONCLUSIONS: These findings suggest that alpha-glutathione-S-transferase levels may be considered a predictive index of response to interferon therapy in chronic hepatitis C patients.

Adult↗
PubMed2000

Cloning of a human tRNA isopentenyl transferase.

A cDNA of human origin is shown to encode a tRNA isopentenyl transferase (E.C. 2.5.1.8). Expression of the gene in a Saccharomyces cerevisiae mutant lacking the endogenous tRNA isopentenyl transferase MOD5 resulted in functional complementation and reintroduction of isopentenyladenosine into tRNA. The deduced amino acid sequence contains a number of regions conserved in known tRNA isopentenyl transferases. The similarity to the S. cerevisiae MOD5 protein is 53%, and to the Escherichia coli MiaA protein 47%. The human sequence was found to contain a single C2H2 Zn-finger-like motif, which was detected also in the MOD5 protein, and several putative tRNA transferases located by BLAST searches, but not in prokaryotic homologues.

Alkyl and Aryl Transferases↗
PubMed2002

Farnesyl transferase inhibitors as anticancer agents.

Protein farnesylation catalysed by the enzyme farnesyl protein transferase involves the addition of a 15-carbon farnesyl group to conserved amino acid residues at the carboxyl terminus of certain proteins. Protein substrates of farnesyl transferase include several G-proteins, which are critical intermediates of cell signalling and cytoskeletal organisation such as Ras, Rho, PxF and lamins A and B. Activated Ras proteins trigger a cascade of phosphorylation events through sequential activation of the PI3 kinase/AKT pathway, which is critical for cell survival, and the Raf/Mek/Erk kinase pathway that has been implicated in cell proliferation. Ras mutations which encode for constitutively activated proteins are found in 30% of human cancers. Because farnesylation of Ras is required for its transforming and proliferative activity, the farnesyl protein transferase inhibitors were designed as anticancer agents to abrogate Ras function. However, current evidence suggests that the anticancer activity of the farnesyl transferase inhibitors may not be simply due to Ras inhibition. This review will discuss available clinical data on three of these agents that are currently undergoing clinical trials.

Alkyl and Aryl Transferases↗
PubMed1996

A new enzyme superfamily - the phosphopantetheinyl transferases.

BACKGROUND: All polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases require posttranslational modification of their constituent acyl carrier protein domain(s) to become catalytically active. The inactive apoproteins are converted to their active holo-forms by posttranslational transfer of the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a conserved serine residue in each acyl carrier protein domain. The first P-pant transferase to be cloned and characterized was the recently reported Escherichia coli enzyme ACPS, responsible for apo to holo conversion of fatty acid synthase. Surprisingly, initial searches of sequence databases did not reveal any proteins with significant peptide sequence similarity with ACPS. RESULTS: Through refinement of sequence alignments that indicated low level similarity with the ACPS peptide sequence, we identified two consensus motifs shared among several potential ACPS homologs. This has led to the identification of a large family of proteins having 12-22 % similarity with ACPS, which are putative P-pant transferases. Three of these proteins, E. coli EntD and o195, and B. subtilis Sfp, have been overproduced, purified and found to have P-pant transferase activity, confirming that the observed low level of sequence homology correctly predicted catalytic function. Three P-pant transferases are now known to be present in E. coli (ACPS, EntD and o195); ACPS and EntD are specific for the activation of fatty acid synthase and enterobactin synthetase, respectively. The apo-protein substrate for o195 has not yet been identified. Sfp is responsible for the activation of the surfactin synthetase. CONCLUSIONS: The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains. This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.

Amino Acid Sequence↗