Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents.

Rat liver microsomes exhibit glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as the second substrate. This activity can be stimulated 8-fold by treatment of the microsomes with N-ethylmaleimide and 4-fold with iodoacetamide. The corresponding glutathione S-transferase activity of the supernatant fraction is not affected by such treatment. These findings suggest that rat liver microsomes contain glutathione S-transferase distinct from those found in the cytoplasmic and that the microsomal transferase can be activated by modification of microsomal sulfhydryl group(s).     

Effect of fraction V powder from bovine serum on rat liver microsomal glutathione S-transferase activity

When bovine serum fraction V powder, which contains 98-99% albumin, is incubated with rat liver microsomes, the glutathione S-transferase activity is stimulated 3-fold. The fraction V stimulates the transferase activity even in microsomes pretreated with N-ethylmaleimide. There is no such stimulating activity in protein fractions other than fraction V. The corresponding cytosolic enzyme activity is not changed by fraction V powder or by N-ethylmaleimide. Neither treatment with charcoal nor heating in boiling water for 5 min can remove the stimulating activity from fraction V.     

Studies of endogenous inhibitors of microsomal glutathione S-transferase.

Glutathione S-transferase is present in rat liver microsomal fraction, but its activity is low relative to the transferase activity present in the soluble fraction of the hepatocyte. We have found, however, that the activity of microsomal glutathione S-transferase is increased 5-fold after treatment with small unilamellar vesicles made from phosphatidylcholine. The increase in activity is due to the removal of an inhibitor of the enzyme from the microsomal membrane. The inhibitor is present in the organic layer of a washed Folch extract of the microsomal fraction. When this fraction of the microsomal extract is reconstituted in the form of small unilamellar vesicles, it inhibits microsomal glutathione S-transferase that had been activated by prior treatment with small unilamellar vesicles of pure phosphatidylcholine, but does not affect the activity of unactivated microsomal glutathione S-transferase. The inhibitor did not seem to be formed during the isolation of the microsomal fraction, and hence may be a physiological regulator of microsomal glutathione S-transferase. In this regard, both free fatty acid (palmitate) and lysophosphatidylcholine were shown to inhibit the enzyme reversibly. The results indicate that the activity of microsomal glutathione S-transferase is far greater than appreciated until now, and that this form of the enzyme may be an important factor in the hepatic metabolism of toxic electrophiles.     

Inhibition of glutathione S-transferase by bile acids.

The effects of bile acids on the detoxification of compounds by glutathione conjugation have been investigated. Bile acids were found to inhibit the total soluble-fraction glutathione S-transferase activity from rat liver, as assayed with four different acceptor substrates. Dihydroxy bile acids were more inhibitory than trihydroxy bile acids, and conjugated bile acids were generally less inhibitory than the parent bile acid. At physiological concentrations of bile acid, the glutathione S-transferase activity in the soluble fraction was inhibited by nearly 50%. This indicates that the size of the hepatic pool of bile acids can influence the ability of the liver to detoxify electrophilic compounds. The A, B and C isoenzymes of glutathione S-transferase were isolated separately. Each was found to be inhibited by bile acids. Kinetic analysis of the inhibition revealed that the bile acids were not competitive inhibitors of either glutathione or acceptor substrate binding. The microsomal glutathione S-transferase from guinea-pig liver was also shown to be inhibited by bile acids. This inhibition, however, showed characteristics of a non-specific detergent-type inhibition.     

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing. Bacterial clones harboring inserts complementary to glutathione S-transferase mRNA were identified by colony hybridization using a [32P]cDNA probe reverse transcribed from poly(A+)-RNA enriched significantly in glutathione S-transferase B mRNA and by hybrid-select translation. Two recombinant clones, pGTB6 and pGTB15 hybrid-selected the mRNAs specific for the Ya and Yc subunits, indicating these two mRNAs share significant sequence homology. Radiolabeled pGTB6 was utilized in RNA gel-blot experiments to determine that the size of glutathione S-transferase B mRNA is 980 nucleotides and the degree of induction of the mRNA in response to 3-methylcholanthrene administration is threefold.     

Studies on the glutathione S-transferase activity associated with rat liver mitochondria.

The major proportion of rat liver glutathione S-transferase is cytosolic. Carefully washed mitochondria contain 0.25-0.47% of the cytosolic activity. Subfractionation of washed mitochondria using digitonin treatment revealed that glutathione S-transferase release did not parallel that of any of the mitochondrial marker enzymes. Glutathione S-transferase release paralleled that of lactate dehydrogenase, suggesting that these 'mitochondrial' activities are due to loosely bound cytoplasmic forms.     

The development of glutathione S-transferase and glutathione peroxidase activities in human lung

The development of glutathione S-transferase and glutathione peroxidase activities has been studied in human lung cytosols. Whilst no clear change in glutathione peroxidase activity was identified, expression of the acidic glutathione S-transferase isoenzyme decreased markedly after 15 weeks of gestation so that at birth the level of activity of this isoenzyme was only about 20% of that in samples obtained during the first trimester. Basic glutathione S-transferase isoenzymes were weakly expressed during development and usually comprised less than 10% of cytosolic activity. Ion-exchange studies identified several basic isoenzymes that may correspond to the alpha, beta, gamma, delta and epsilon set previously identified in liver. Weak expression of apparently near-neutral isoenzymes was also detected; they were detected in only a few cytosols.     

The requirement for glutathione S-transferase in the conjugation of activated aflatoxin B1 during aflatoxin hepatocarcinogenesis in the rat

The formation of an aflatoxin B1-reduced glutathione (AFB1-GSH) conjugate in in vitro systems has been examined. AFB1 was activated by a chicken liver microsomal system and factors affecting the subsequent conversion to the AFB1-dihydrodiol or conjugation with GSH were investigated by HPLC. A requirement for glutathione S-transferase in the formation of the AFB1-GSH conjugate was observed. Studies using CM-cellulose columns showed the fractions containing glutathione S-transferase B activity were the most effective in catalysing the formation of the AFB1-GSH conjugate. The possibility of changes in the level of AFB1-GSH conjugate production in the liver during carcinogenesis by AFB1 has been examined. It has been found, using freshly isolated rat hepatocytes, that low level feeding with AFB1 in vivo increases the production of the conjugate in vitro. Further increases in the production of the conjugate by hepatocytes in vitro, accompanying increases in the preneoplastic lesions, are achieved by partially hepatectomising the AFB1-fed animals. Partial hepatectomy of control-fed animals yielded no similar changes. The AFB1/partial hepatectomy treatment resulted in increased levels of all the glutathione S-transferase activities fractionated on CM-cellulose. Macromolecular binding of AFB1 and/or of its metabolites was detected in the fractions containing glutathione S-transferase activity, but there was no evidence for a greater binding in the glutathione S-transferase B/ligandin containing fractions. Furthermore fractionation on Sephadex G-75 indicated a predominance of binding of AFB1 to proteins of a higher molecular weight than the glutathione S-transferases, although some binding in the molecular weight range of the latter was observed.     

Human microsomal glutathione S-transferase. Its involvement in the conjugation of hexachlorobuta-1,3-diene with glutathione.

A microsomal glutathione S-transferase (GST) was purified from human liver. This enzyme was shown to have characteristics similar to those of the rat microsomal GST described by Morgenstern & De Pierre [(1983) Eur. J. Biochem. 134, 591-597]. The specific activity of human microsomal GST towards 1-chloro-2,4-dinitrobenzene or cumene hydroperoxide can be stimulated by treating the enzyme with N-ethylmaleimide. This enhancement of activity is accompanied by increased sensitivity to inhibition by haematin and cholic acid. The subunit Mr values of the rat and human enzymes are similar (approx. 17,300), and the proteins are immunologically related. During purification, both human and rat microsomal GST enzymes are the only hepatic proteins obtained from Triton X-100-solubilized microsomal fractions that show activity towards the nephrotoxin hexachlorobuta-1,3-diene. The involvement of microsomal GST in toxification reactions is discussed.     

Stereochemistry of the microsomal glutathione S-transferase catalyzed addition of glutathione to chlorotrifluoroethene

The stereochemistry of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation was studied in rat liver cytosol, microsomes, N-ethylmaleimide-treated microsomes, 9000g supernatant fractions, purified rat liver microsomal glutathione S-transferase, and isolated rat hepatocytes. The absolute configuration of the chiral center generated by the addition of glutathione to chlorotrifluoroethene was determined by degradation of S-(2-chloro-1,1,2-trifluoroethyl)glutathione to chlorofluoroacetic acid, followed by derivatization to form the diastereomeric amides N-(S)-alpha-methylbenzyl-(S)-chlorofluoacetamide and N-(S)-alpha-methylbenzyl-(R)-chlorofluoroacetamide, which were separated by gas chromatography. Native and N-ethylmaleimide-treated rat liver microsomes, purified rat liver microsomal glutathione S-transferase, rat liver 9000g supernatant, and isolated rat hepatocytes catalyzed the formation of 75-81% (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione; rat liver cytosol catalyzed the formation of equal amounts of (2R)- and (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione. In rat hepatocytes, microsomal glutathione S-transferase catalyzed the formation of 83% of the total S-(2-chloro-1,1,2-trifluoroethyl)glutathione formed. These observations show that the microsomal glutathione S-transferase catalyzes the first step in the intracellular, glutathione-dependent bioactivation of the nephrotoxin chlorotrifluoroethene.     

Developmental aspects of glutathione S-transferase B (ligandin) in rat liver.

The postnatal development in male Sprague-Dawley rats of hepatic glutathione S-transferase B (ligandin) in relation to the other glutathione S-transferases is described. The concentration of glutathione S-transferase B in 1-day-old male rats is about one-fifth of that in adult animals. The enzyme reaches adult concentrations 4-5 weeks later. When assessed by substrate specificity or immunologically, the proportion of transferase B relative to the other glutathione S-transferases is high during the first week after birth. At this age, 67.5% of the transferase activity towards 1-chloro-2,4-dinitrobenzene is immunoprecipitable by anti-(transferase B), compared with about 50% in adults and older pups. Between the second and the fifth postnatal week, the fraction of transferase B increases in parallel fashion with the other transferases in hepatic cytosol. Neither L-thyroxine nor cortisol induce a precocious increase in glutathione S-transferase activity. Phenobarbital did induce transferase activity towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene in both pups and adults. The extent of induction by phenobarbital was a function of basal activity during development such that the percentage stimulation remained constant from 5 days postnatally to adulthood.     

Characterization of glutathione S-transferase of Taenia solium.

A Taenia solium glutathione-S-transferase fraction (SGSTF) was isolated from a metacestode crude extract by affinity chromatography on reduced glutathione (GSH)-sepharose. The purified fraction displayed a specific glutathione S-transferase (GST) activity of 2.8 micromol/min/mg and glutathione peroxidase selenium-independent activity of 0.22 micromol/min/mg. Enzymatic characterization of the fraction suggested that the activity was closer to the mammalian mu-class GSTs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and enzyme activity analysis showed that the fraction was composed of a major band of Mr = 26 kd and that the active enzyme was dimeric. Immunohistochemical studies using specific antibodies against the major 26-kd band of the SGSTF indicated that GST protein was present in the tegument, parenchyma, protonephridial, and tegumentary cytons of the T. solium metacestode. Antibodies generated against the SGSTF tested in western blot showed cross-reactivity against GSTs purified from Taenia saginata, T. taeniaeformis, and T. crassiceps, but did not react with GSTs from Schistosoma mansoni, or mice, rabbit, and pig liver tissue. Furthermore, immunization of mice with SGSTF reduced the metacestode burden up to 74.2%. Our findings argue in favor of GST having an important role in the survival of T. solium in its hosts.     

Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.     

Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species

The effect of enzymatically generated reduced oxygen metabolites on the activity of hepatic microsomal glutathione S-transferase activity was studied to explore possible physiological regulatory mechanisms of the enzyme. Noradrenaline and the microsomal cytochrome P-450-dependent monooxygenase system were used to generate reduced oxygen species. When noradrenaline (greater than 0.1 mM) was incubated with rat liver microsomes in phosphate buffer (pH 7.4), an increase in microsomal glutathione S-transferase activity was observed, and this activation was potentiated in the presence of a NADPH-generating system; the glutathione S-transferase activity was increased to 180% of the control with 1 mM noradrenaline and to 400% with both noradrenaline and NADPH. Superoxide dismutase and catalase inhibited partially the noradrenaline-dependent activation of the enzyme. In the presence of dithiothreitol and glutathione, the activation of the glutathione S-transferase by noradrenaline, with or without NADPH, was not observed. In addition, the activation of glutathione S-transferase activity by noradrenaline and glutathione disulfide was not additive when both compounds were incubated together. These results indicate that the microsomal glutathione S-transferase is activated by reduced oxygen species, such as superoxide anion and hydrogen peroxide. Thus, metabolic processes that generate high concentrations of reduced oxygen species may activate the microsomal glutathione S-transferase, presumably by the oxidation of the sulfhydryl group of the enzyme, and this increased catalytic activity may help protect cells from oxidant-induced damage.     

Increase in glutathione disulfide level regulates the activity of microsomal glutathione S-transferase in rat liver

Glutathione disulfide stimulates the activity of rat liver microsomal glutathione S-transferase 2-fold after incubation at 25 degrees C for 10 min. When the microsomes were incubated with the disulfide for over 20 min, the transferase activity increased to the same extent as in the case of N-ethylmaleimide (6-fold). Even in the presence of reduced glutathione, some enhancement of the transferase activity was observed. The data presented here are evidence that increase in glutathione disulfide level, e.g. by lipid peroxidation, on endoplasmic reticulum causes the upregulation of microsomal glutathione S-transferase activity.     

The distribution and comparison of glutathione, glutathione reductase and glutathione S-transferase in various camel tissues

Extracts prepared from liver, kidney, lung and brain of camel contain glutathione, glutathione S-transferase and glutathione reductase. Liver had the highest level of glutathione (218.7 mumol/g wet weight) whereas brain had the lowest level (66.4 mumol/g wet weight). The highest activity for glutathione reductase was found in the kidney (2.6 mumol/min/mg protein) while the lowest activity was found in the lung (0.9 mumol/min/mg protein). Glutathione S-transferase activity was the highest in liver (4.2 mumol/min/mg protein) and the lowest in brain (1 mumol/min/mg protein). Purified glutathione S-transferases from lung, kidney, brain and liver were similar in their molecular size, subunit composition as well as immuno-reactivity and showed some differences in their response to heat and inhibitors.     

Camel brain glutathione S-transferase purification, properties, regional and subcellular distribution

1. Camel brain glutathione S-transferase was purified by glutathione-linked agarose affinity column and the different isozymes were separated by chromatofocusing. 2. The basic isozymes which comprise 45% of the total activity were immunologically indistinguishable from the near-neutral isozymes which constitute 55% of the activity. 3. Some differences were detectable among the basic and near-neutral isozymes in relation to substrate specificities and subunit composition. 4. Biochemical and immunological quantification of glutathione S-transferase revealed the presence of the enzyme in all camel brain regions tested and subcellular fractions. 5. The pons had the highest concentration of the enzyme and the cortex had the lowest, while more than 88% of the enzyme was present in the cytosol.     

Studies on human placental glutathione S-transferase. Multiplicity and mother age influence.

Human placental glutathione S-transferase was purified to apparent homogeneity by direct application of the crude homogenate into glutathione linked sepharose affinity chromatography. Chromatofocusing analysis in the presence of reduced glutathione resolved the enzyme into three acidic peaks eluted at pH 6.0, 5.7 and 5.5. About 36% of the initial activity was recovered in the isozyme fraction eluted at pH 6.0 whereas the isozymes eluted at pH 5.7 and 5.5 accounted for 20% and 25% of the activity respectively. Disc gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a single protein band in all the three separated isozymes. These isozymes were homodimers with an apparent relative molecular mass of 44.000 and subunit molecular mass of 21.000. The isozymes were immunologically related to each other and to the enzyme from goat and sheep placentae. Mother age had no influence in the placental glutathione S-transferase activity, albeit the activity was slightly higher in placenta obtained from younger women.     

The hepatic glutathione content and glutathione S-transferase activity in the pike (Esox lucius L.) and rat.

1. The content of glutathione and glutathione disulfide and the activity of the glutathione S-transferase were determined in the liver of pike and rat. 2. It was found that the liver of pike contains far less glutathione than the liver of rats, while the glutathione disulfide content was similar in both species. 3. The activity of the hepatic glutathione S-transferase was more effective in pike than in rats.     

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.