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PubMed1993

Glutathione S-transferases in esophageal cancer.

Glutathione content and glutathione S-transferase enzyme activity as well as isoenzyme composition were studied in normal gastric cardia, normal squamous esophageal epithelium and corresponding malignant tumor of 10 patients with esophageal cancer. Mean values of glutathione (38 +/- 6 versus 36 +/- 12 nmol/mg protein) and glutathione S-transferase activity (532 +/- 44 versus 532 +/- 108 nmol/min mg protein) did not differ significantly between normal esophageal and tumor tissue. However, great individual differences exist. In two patients, glutathione S-transferase activity was much higher in the tumor (1081 and 1381 nmol/min mg protein) due to overexpression of class alpha, mu and pig glutathione S-transferases in one case, and of class mu and pi in the other case. In the other patients, glutathione S-transferase activity was equal (one case) or lower (seven cases) in the tumor. In normal gastric cardia glutathione content as well as glutathione S-transferase activity was significantly lower as compared to normal esophageal epithelium. In conclusion, in contrast to other gastrointestinal tumors, glutathione S-transferases are overexpressed in esophageal tumors in only a limited number of patients.

Adult↗
PubMed1984

Quantitative significance of glutathione and glutathione-S-transferase in regulating benzo[a]pyrene anti diol-epoxide level in reconstituted C3H/10T1/2 cell lysates, and comparison to rat liver.

Induced 10T1/2 cell microsomes were independently reconstituted with [3H]benzo[a]pyrene (BP) and glutathione (GSH) or purified GSH-transferases. Levels of the primary BP anti 7,8-dihydrodiol 9,10-epoxide (r-7,t-8 dihydroxy-t-9,10-oxy-7,8,9,10 tetrahydrobenzo[a]pyrene) hydrolysis product, 7,10/8,9-tetrol, were measured in incubation extracts, enabling us to monitor the level of free anti diol-epoxide in incubations and to determine the independent effects of GSH or GSH-transferases upon it. GSH alone had no effect on anti diol-epoxide levels over the concentration range tested (0-4.0 mM), however, the addition of purified GSH-transferase from rat liver resulted in a dose-dependent conjugation of anti diol-epoxide as well as 9,10-epoxide and 7,8-epoxide with 50% conjugation occurring at 0.036, 0.039 and 0.17 units GSH-transferase/ml, respectively. Free anti diol-epoxide was reduced by greater than 95% when we reconstituted with the GSH-transferase concentration which we measured in 10T1/2 cells (0.15-0.27 units/ml cell cytosol); this GSH-transferase concentration represents only 6% of that found in rat liver. The results suggest that in both 10T1/2 cells and rat hepatocytes GSH-transferase catalyzed GSH conjugation is quantitatively significant in determining the intracellular level of anti diol-epoxide.

Animals↗
PubMed1985

Hereditary interindividual differences in the glutathione transferase activity towards trans-stilbene oxide in resting human mononuclear leukocytes are due to a particular isozyme(s).

We have shown earlier that there are large, hereditary interindividual differences in the cytosolic glutathione transferase activity towards trans-stilbene oxide in human mononuclear leukocytes. In the present study we ask whether these differences reflect the presence or absence of a particular isozyme(s) of glutathione transferase. First, in order to measure the high glutathione transferase activity optimally it was necessary to modify our previous assay by increasing the concentration of reduced glutathione from 3 to 5 mM and of the substrate from 50 to 250 microM. It was then found that the low activity demonstrates an apparent Km for trans-stilbene oxide of 28.3 microM, whereas the corresponding value for the high activity was 127 microM. Secondly, it was found that while glutathione transferase activity towards trans-stilbene oxide in different individuals segregated into three groups, low, high and very high, glutathione transferase activity towards 1-chloro-2,4-dinitrobenzene in these same mononuclear leukocyte fractions formed only a single group with no tendency towards such segregation. Thirdly, the SDS-poly-acrylamide gel electrophoretic pattern obtained with the supernatant fraction from mononuclear cells demonstrating high glutathione transferase activity towards trans-stilbene oxide contained a band of 25 000 molecular weight which was either absent or present at a much lower level in cells demonstrating low activity. We conclude that high activity towards trans-stilbene oxide in circulating, resting human mononuclear cells is catalyzed by a particular isozyme(s) of glutathione transferase. cis-Stilbene oxide, styrene oxide and, possibly, benzo[a]pyrene 4,5-oxide are also substrates for this isozyme(s).

Dinitrochlorobenzene↗
PubMed1993

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

The amino acid sequence of glutathione S-transferase a from guinea pig liver was determined. Glutathione S-transferase a was composed of two identical subunits, each comprising 218 amino acid residues. The amino acid sequence of glutathione S-transferase a exhibited 73% homology with that of human glutathione S-transferase Ha, 69% with that of rat glutathione S-transferase Ya, and 68% with that of rat glutathione S-transferase Yc, which are known to belong to class Alpha. From the above result, together with previous observations on its substrate specificity, it was concluded that glutathione S-transferase a belonged to class Alpha.

Amino Acid Sequence↗
PubMed2006

A novel class of CoA-transferase involved in short-chain fatty acid metabolism in butyrate-producing human colonic bacteria.

Bacterial butyryl-CoA CoA-transferase activity plays a key role in butyrate formation in the human colon, but the enzyme and corresponding gene responsible for this activity have not previously been identified. A novel CoA-transferase gene is described from the colonic bacterium Roseburia sp. A2-183, with similarity to acetyl-CoA hydrolase as well as 4-hydroxybutyrate CoA-transferase sequences. The gene product, overexpressed in an Escherichia coli lysate, showed activity with butyryl-CoA and to a lesser degree propionyl-CoA in the presence of acetate. Butyrate, propionate, isobutyrate and valerate competed with acetate as the co-substrate. Despite the sequence similarity to 4-hydroxybutyrate CoA-transferases, 4-hydroxybutyrate did not compete with acetate as the co-substrate. Thus the CoA-transferase preferentially uses butyryl-CoA as substrate. Similar genes were identified in other butyrate-producing human gut bacteria from clostridial clusters IV and XIVa, while other candidate CoA-transferases for butyrate formation could not be detected in Roseburia sp. A2-183. This suggests strongly that the newly identified group of CoA-transferases described here plays a key role in butyrate formation in the human colon.

Acyl Coenzyme A↗
PubMed1983

Microsomal glutathione transferase. Purification in unactivated form and further characterization of the activation process, substrate specificity and amino acid composition.

The procedure developed for purification of the N-ethylmaleimide-activated microsomal glutathione transferase was applied successfully to isolation of this same enzyme in unactivated form. The microsomal glutathione transferases, the unactivated and activated forms, were shown to be identical in terms of molecular weight, immunochemical properties, and amino acid composition. In addition the microsomal glutathione transferase purified in unactivated form could be activated 15-fold with N-ethylmaleimide to give the same specific activity with 1-chloro-2,4-dinitrobenzene as that observed for the enzyme isolated in activated form. This activation involved the binding of one molecule N-ethylmaleimide to the single cysteine residue present in each polypeptide chain of the enzyme, as shown by amino acid analysis, determination of sulfhydryl groups by 2,2'-dithiopyridyl and binding of radioactive N-ethylmaleimide. Except for the presence of only a single cysteine residue and the total absence of tryptophan, the amino acid composition of the microsomal glutathione transferase is not remarkable. The contents of aspartic acid/asparagine + glutamic acid/glutamine, of basic amino acids, and of hydrophobic amino acids are 15%, 12% and 54% respectively. The isoelectric point of the enzyme is 10.1. Microsomal glutathione transferase conjugates a wide range of substrates with glutathione and also demonstrates glutathione peroxidase activity with cumene hydroperoxide, suggesting that it may be involved in preventing lipid peroxidation. Of the nine substrates identified here, the enzymatic activity towards only two, 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide, could be increased by treatment with N-ethylmaleimide. This treatment results in increases in both the apparent Km values and V values for 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Thus, although clearly distinct from the cytosolic glutathione transferases, the microsomal enzyme shares certain properties with these soluble enzymes, including a relative abundance, a high isoelectric point and a broad substrate specificity. The exact role of the microsomal glutathione transferase in drug metabolism, as well as other possible functions, remains to be established.

Amino Acids↗
PubMed1975

Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii.

Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii has been purified and crystallized, and some of its properties have been established. The work was facilitated by a newly developed coupled and continuous spectrophotometric assay in which the disappearance of added acrylate could be followed at 245 nm. The rate-limiting conversion of acetyl- and beta-hydroxypropionyl CoA to acrylyl CoA by CoA transferase was followed by the non-rate-limiting conversion to beta-hydroxypropionyl CoA by excess crotonase. Thus, a small priming quantity of acetyl CoA served to generate acrylyl CoA, which, by hydration, generated beta-hydroxypropionyl CoA. This product then served to generate more acrylyl CoA in cyclic fashion. The net result was the CoA transferase-limited conversion of acrylate to beta-hydroxypropionate. The purified transferase has a molecular weight of 125,000 and is composed of two subunits of 63,000 each, as determined by disc gel electrophoresis. Short-chain-length monocarboxylic acids are substrates, whereas dicarboxylic or beta-ketocarboxylic acids are not. The reaction kinetics are typical of a ping-pong bi bi mechanism composed of two half reactions linked by a covalent enzyme intermediate. Incubation of the transferase with acetyl CoA in the absence of a fatty acid acceptor yielded a stable intermediate which, by absorption spectrophotometry, radioactivity measurements, reduction with borohydride, reactivity with hydroxylamine, and catalytic activity, was identified as an enzyme-CoA compound. Kinetic constants for CoA transferase are: final specific activity, 110 U/mg of protein corresponding to 1.38 X 10(4) mumol of acrylate activated per mumol of transferase; Km for acrylate, 1.2 X 10(-3) M; Km for acetyl CoA (beta-hydroxypropionyl CoA), 2.4 X 10(-5) M.

Acetyl Coenzyme A↗
PubMed1992

Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida.

beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified.

Adipates↗
PubMed1989

Effects of 2(3)-tert-butyl-4-hydroxyanisole pretreatment on cefpiramide binding to mouse glutathione S-transferases.

Binding of cefpiramide (CPM) and other beta-lactam antimicrobial agents to 2(3)-tert-butyl-4-hydroxyanisole (BHA)-induced liver glutathione (GSH) S-transferases (EC 2.5.1.18) from CD-1 mice was studied. A marked induction of hepatic GSH S-transferase from mice fed BHA was observed. Gel chromatography of liver cytosol from mice fed BHA showed an increased binding of CPM, cefotetan and cefazolin to BHA-induced GSH S-transferases. The extent of their binding to GSH S-transferase seemed to be correlated with the extent of their excretion into the bile. Binding of CPM to the GSH S-transferase fraction was inhibited by both indocyanine green, which is known to bind liver GSH S-transferases intensively, and by cefoperazon, which is mainly excreted into the bile. This study suggests that GSH S-transferases are the main binding proteins of CPM in the liver cytosol fraction and play an important role as carrier proteins of CPM and some antimicrobial agents in mouse liver.

Animals↗
PubMed1994

Urinary pi-class glutathione transferase as an indicator of tubular damage in the human kidney.

Glutathione transferase-pi released from kidney tubular epithelial cells was analyzed in the urine of recipients of renal allografts. Urinary content of alpha-class glutathione transferase was also determined for comparison. Control urine from healthy individuals contained detectable levels of the pi-isoenzyme (6.6 +/- 0.46 ng/ml, mean +/- SEM) and this concentration was not increased in the urine of patients demonstrating cyclosporine A-induced nephrotoxicity (6.3 +/- 0.29 ng/ml), in contrast to the alpha-form. Acute rejection increased excretion of the pi-isoenzyme (19.0 +/- 2.0 ng/ml), but not of the alpha-glutathione transferase. Thus, while the serum creatinine level increases in connection with both cyclosporine A-induced nephrotoxicity and acute rejection, analyses of urinary glutathione transferases distinguish well between these conditions. Acute tubular necrosis and renal transplant infarction resulted in a rapid elevation in urinary levels of both alpha- and pi-transferase. The advantages of this approach are that release of the protein into the urine occurs rapidly after tubular damage, the assay is sensitive and specific and can also distinguish between certain pathological conditions. These studies thus indicate that the urinary level of glutathione transferase-pi can be used for monitoring certain pathological processes in the kidney. Quantitation of this enzyme complements the information obtained by measurement of glutathione transferase-alpha.

Adolescent↗
PubMed1992

Activation of hepatic microsomal glutathione S-transferase of rats by a glutathione depletor, diethylmaleate.

The effect of glutathione depletor diethylmaleate on rat hepatic glutathione S-transferase and glutathione peroxidase was studied in vivo and in vitro. When diethylmaleate (600 mg/kg) was given i.p. to rats, liver glutathione was depleted within 2 h and recovered to the control level 5 h after diethylmaleate treatment. Both glutathione S-transferase and peroxidase activities in microsomes, not in cytosol, were markedly increased during glutathione depletion and only glutathione S-transferase activity remained at high levels after recovery of the glutathione content. The increase in microsomal glutathione S-transferase and peroxidase activities with concomitant exhaustion of glutathione was also observed by perfusion of the isolated liver with diethylmaleate (10 mM). When liver microsomes were incubated with diethylmaleate in vitro at 37 degrees C, glutathione S-transferase, but not peroxidase, activity was increased; the increase was not reversed by dithiothreitol. These results indicate that diethylmaleate activates microsomal glutathione S-transferase by direct reaction to the enzyme during glutathione depletion and suggest that glutathione S-transferase activity and glutathione peroxidase activity in the microsomal enzyme may be differently regulated.

Animals↗
PubMed1984

Differential determination of cationic and anionic glutathione S-transferases by enzyme immunoassay.

Specific enzyme immunoassays for cationic and anionic glutathione S-transferases were established using the specific antibodies which were purified by antigen-bound adsorbent column chromatography. The enzyme immunoassay for cationic glutathione S-transferase had high specificity to cationic enzyme, but showed no cross reactivity with anionic one, and vice versa. The recovery of cationic glutathione S-transferase by the enzyme immunoassay was 94.7%, and coefficient of variation for within day and day-to-day precision were 7.8-10.4% and 8.5-12.5%, respectively. The enzyme immunoassay for anionic glutathione S-transferase also had a good recovery and precision. Using these enzyme immunoassays for glutathione S-transferases, sera of various patients were analyzed. Serum cationic glutathione S-transferase was increased in patients with hepatitis and hepatoma, and anionic glutathione S-transferase in serum was increased in patients with liver cirrhosis.

Animals↗
PubMed1989

Glutathione S-transferases and chloroform toxicity in streptozotocin-induced diabetic rats.

The glutathione S-transferase activity in liver and kidney cytosol was significantly decreased in short term diabetes induced with streptozotocin, whereas no decrease in the transferase was observed in phenobarbital-treated diabetic rats. Toxicity of chloroform was potentiated in streptozotocin- or phenobarbital-treated rats. The decrease in liver cytosolic and microsomal glutathione S-transferase activity was observed in long term diabetic rats, and only microsomal transferase activity was restored by insulin treatment. There was no release of glutathione S-transferases into the serum in the diabetic rats, and the transferases were not inhibited by streptozotocin in vitro. These results showed that glutathione S-transferase activity decreased during diabetes, and this decrease may contribute to altering drug metabolism and toxicity in diabetes.

Alanine Transaminase↗
PubMed1994

Alteration of liver glutathione S-transferase and protease activities by cobalt chloride treatment of rats.

Effects of cobalt chloride on liver glutathione S-transferase and protease activities were studied. When cobalt chloride (60 mg/kg) was given to rats, liver microsomal glutathione S-transferase and protease activities were significantly increased 24 hr after the injection, whereas glutathione peroxidase activity in microsomes was decreased. The increase in glutathione S-transferase by N-ethylmaleimide was similar to that of the control, indicating that the increase in the transferase activity by cobalt chloride is not due to a modification of the sulfhydryl group of the enzyme. Immunochemical analysis of the liver microsomes did not detect any proteolytic product of microsomal glutathione S-transferase. In puromycin- or actinomycin D-treated rats, an increase in the transferase activity caused by cobalt chloride treatment was depressed. Thus it was suggested that liver microsomal glutathione S-transferase is induced by cobalt chloride treatment, but not activated by limited proteolysis via microsomal protease.

Animals↗
PubMed1980

Resolution, purification and some properties of three glutathione transferases from rat liver mitochondria.

From the matrix of rat liver mitochondria, three GSH transferases were isolated and named Transferase 1, 2.1 and 2.2. Transferases 1 and 2.2 were purified to electrophoretic homogeneity. Transferase 1 contributes up to about 90% of the total mitochondrial GSH-transferring activity. It has a molecular weight of approx. 45 000 and is composed of two subunits of similar size. The isoelectric point is at pH 7.1-7.4. The Km values for GSH and 1-chloro-2,4-dinitrobenzene are 0.3 and 0.7 mmol/l respectively. Transferase 2.2 has the same molecular weight and subunit structure like the Transferase 1 and an isoelectric point at pH 4.8. The apparent Km values for GSH and 1-chloro-2,4-dinitrobenzene are 0.3 mmol/l and 0.4 mmol/l respectively. Transferase 2.1 contributes only 1% of the total mitochondrial GSH-transferring activity. It has high apparent Km values for both GSH and 1-chloro-2,4-dinitrobenzene (5.6 mmol/l and 1.3 mmol/l resp.) and a limited spectrum of substrates.

Animals↗
PubMed1981

Purification and characterization of an acid glutathione S-transferase from human lung.

An acid glutathione S-transferase (EC: 2.5.1.18) from human lung was purified and characterized. The purification procedure included two isoelectric focusing runs, Sephadex G-100 gel filtration, glutathione-affinity chromatography, and Sephadex G-75 gel filtration. With respect to the properties studied the acid lung transferase differed from human liver transferases alpha-epsilon, but it bore a close resemblance to the other human low pI transferases. Bilirubin affected the kinetics of the lung enzyme markedly differently as compared with transferase p, suggesting possible nonidentity between these enzymes. The acid lung transferase represented about 97% of the total glutathione transferase activity of the lung 100,000 g supernatant used in this work.

Chromatography, Affinity↗
PubMed2001

Drug resistance in epithelial ovarian cancer: P-glycoprotein and glutation S-transferase. Can they play an important role in detecting response to platinum-based chemotherapy as a first-line therapy.

OBJECTIVE: Drug resistance is important for the treatment of ovarian cancer. P-glycoprotein and glutation S-transferase as resistance markers play an important role in the effectivity of chemotherapeutical agents. The role of P-glycoprotein and glutation S-transferase in the treatment of epithelial ovarian cancer is not well understood. We investigated the relation between P-glycoprotein and glutation S-transferase level for response to platinum-based chemotherapy in epithelial ovarian cancer. MATERIAL AND METHODS: We reviewed 30 cases diagnosed as epithelial ovarian cancer and treated with platinum-based chemotherapy in the Department of Obstetrics and Gynecology, Akdeniz University School of Medicine. The material was attained from initial parafin-embeded blocks stained for P-glycoprotein and glutation S-transferase. The cases that were diagnosed and treated before attending our clinic were not enrolled in the study. RESULTS: Mean age was 58.2 (25-70) and mean gravida 4.1 (0-10). Twenty-four patients (80%) were glutation S-transferase positive. Three cases (10%) out of 30 had positive reaction for P-glycoprotein. No difference was revealed regarding chemotherapy response rate among the cases showing glutation S-transferase positivity and P-glycoprotein negativity. CONCLUSION: Detection of glutation S-transferase and P-glycoprotein levels in epithelial ovarian cancer tissue is not important for response to platinum-based chemotherapy as a first line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Inactivation of the genotoxic aldehyde acrolein by human glutathione transferases of classes alpha, mu, and pi.

Acrolein, a genotoxic aldehyde released in the metabolic activation of the cytostatic drug cyclophosphamide, is inactivated by glutathione transferases either by conjugation with reduced glutathione or by covalent binding to the enzymes in the absence of glutathione. The catalytic efficiency (kcat/Km) with acrolein as a substrate was determined for representatives of the three classes Alpha, Mu, and Pi of human glutathione transferases. Transferase pi exhibited the highest and transferase epsilon the lowest catalytic efficiencies, respectively. As measured by the kcat/Km value, acrolein ranks among the most active substrates known for transferase pi. The irreversible binding of acrolein to the enzymes was monitored as the inactivation of the enzyme activity. Transferase pi reacted significantly more rapidly with acrolein than did transferases mu and epsilon.

Acrolein↗