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.     

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.     

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.     

Reduced glutathione inhibits the alkylation by N-nitrosodimethylamine of liver DNA in vivo and microsomal fraction in vitro

The transfer of radioactivity from N-nitroso-[14C]dimethylamine to trichloroacetic acid precipitable macromolecules in the microsomal fraction of rat liver was investigated. This transfer was found to depend on N-nitrosodimethylamine being metabolized. Cytosolic fraction and cytosol enriched with reduced glutathione inhibited the binding of radioactivity to acid insoluble proteins. Depletion of glutathione in rat liver with diethylmaleate prior to i.v. administration of 10 mg N-nitroso-[14C]dimethylamine/kg led to an increase in O6-methylguanine and N-7-methylguanine in DNA. If rats were fed disulfiram for 6 days (2 g/kg feed), glutathione and glutathione S-transferase were enhanced, and the degree of methylation of guanine by N-nitrosodimethylamine was greatly reduced, as was the metabolism of N-nitrosodimethylamine in the intact animal. Fasting rats for 24 h did not change the N-nitrosodimethylamine-demethylase activity in vitro but greatly enhanced the methylation of guanine in vivo, while the glutathione content and glutathione S-transferase activity were not changed compared to fed animals.     

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.     

Re-evaluation of protein-bound glutathione in rat liver

The extent of intracellular glutathione binding to proteins through a disulfide linkage in rat liver was examined quantitatively. The content of glutathione associated with the acid-precipitable fraction and releasable on borohydride treatment was 0.024 +/- 0.016 mumol/g liver, which accounted for less than one per cent of the total glutathione (6-7 mumol/g liver) in the liver of fed rats. Most of the thiol (2-4 mumol/g liver) liberated from liver proteins into the acid-soluble fraction on borohydride reduction in the presence of guanidine hydrochloride was not glutathione but was proteinaceous in nature. The amounts of thiols liberated per g of liver were similar in fed, fasted, and dibutyryl-3',5'-cyclic AMP-treated rats.     

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.     

A comparison of the glutathione S-transferases of trout and rat liver.

1. Cytosol from trout liver, gills and intestinal caeca has substantial glutathione S-transferase activity. 2. Gel-exclusion and ion-exchange chromatography suggest that trout liver has several glutathione S-transferases with different molecular weights and ionic charges. 3. A component capable of binding lithocholic acid eluted together with glutathione S-transferase activity. Some of the transferase activity did not elute together with binding activity. 4. The enzymic activity from trout liver was less stable at 37 degrees C than that from rat liver. 5. The glutathione S-transferases of fish liver have a similar specific activity to those of rat liver but different molecular properties.     

Possible role of blood insulin levels on glutathione S-transferase activities from different tissues of male rats

The activity of in vitro glutathione S-transferase towards 1-chloro-2,4-dinitrobenzene was examined in liver, renal cortex, and small intestine (duodenum, jejunum, ileum) after the in vivo treatment of male Wistar rats with streptozotocin or alloxan. The studies were performed at 2, 10, 24, and 48 h and 7 and 15 days after streptozotocin treatment or 24 and 48 h after alloxan treatment. The results indicated that while the blood levels of insulin-glucose did not show variations, there were no alterations of the glutathione S-transferase activity in the tissues tested. On the other hand, when the treatments caused modifications on blood insulin-glucose levels, there were changes of glutathione S-transferase activity in all tissues (except in the ileum) in such a way that a direct relationship between plasma insulin levels and glutathione S-transferase activity could be demonstrated. These results were also confirmed through insulin administration to control and diabetic rats. The data demonstrate a possible regulation of glutathione S-transferase activity by blood insulin and (or) glucose levels in the tissues tested.     

Subcellular distribution of N-ethylmaleimide-stimulatable glutathione S-transferase activity in rat liver. Evidence of localization of glutathione S-transferase in peroxisomal membrane

Subcellular distribution of glutathione S-transferase activity was investigated as stimulated form by N-ethylmaleimide in rat liver. The stimulated glutathione S-transferase activity was localized in mitochondrial and lysosomal fractions besides microsomes. Among N-ethylmaleimide-treated submitochondrial fractions, glutathione S-transferase activity was stimulated only in outer mitochondrial membrane fraction. In lysosomal fraction, it was suggested that glutathione S-transferase activity in peroxisomes, which is immunochemically related to microsomal transferase, was also stimulated, but not in lysosomes.     

The effects of in vitro lipid peroxidation on the activity of rat liver microsomal glutathione S-transferase from rats supplemented or deficient in antioxidants

Glutathione S-transferases are a group of multifunctional isozymes that play a central role in the detoxification of hydrophobic xenobiotics with electrophilic centers (1). In this study we investigated the effects of in vitro lipid peroxidation on the activity of liver microsomal glutathione S-transferases from rats either supplemented or deficient in both vitamin E and selenium. Increased formation of malondialdehyde (MDA), a by-product of lipid peroxidation, was associated with a decreased activity of rat liver microsomal glutathione S-transferase. The inhibition of glutathione S-transferase occurred rapidly in microsomes from rats fed a diet deficient in both vitamin E and selenium (the B diet) but was delayed for 15 minutes in microsomes from rats fed the same diet but supplemented with these micro-nutrients (B+E+Se diet). Lipid peroxidation inhibits microsomal glutathione S-transferase and this inhibition is modulated by dietary antioxidants.     

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.     

Renal glutathione homeostasis in compensatory renal growth

Glutathione homeostasis was investigated in unilaterally nephrectomized and sham-operated rats. Following twelve days of compensatory renal growth, it was found that the concentrations of glutathione and glutathione disulfide in representative samples of the entire remnant right kidney from the nephrectomized rats were similar to those found in corresponding samples of the right kidneys from the sham-operated rats. However, since the mass of the remnant right kidneys in the nephrectomized rats was greater than that of the right kidneys from the sham-operated rats, the absolute content of glutathione and glutathione disulfide was greater in the remnant right kidneys of the nephrectomized rats than in the right kidneys of the sham-operated rats. In general, the findings from the present study indicate that the absolute content of glutathione and glutathione disulfide in renal epithelial cells increases in proportion to the increase in mass that results from compensatory renal cellular hypertrophy.     

The effects of gonadectomy on the hepatic activities of aryl hydrocarbon hydroxylase, epoxide hydratase, and glutathione S-transferase in Wistar rats pretreated with oral methadone . HCl.

Methadone . HCl given in the drinking water for 4 weeks increased microsomal epoxide hydratase activity in the liver of adult male Wistar rats, with no change in aryl hydrocarbon hydroxylase activity. In contrast, in female rats it raised aryl hydrocarbon hydroxylase with no change in epoxide hydratase activity. Gonadectomy altered the effect of methadone on epoxide hydratase, but not on aryl hydrocarbon hydroxylase activity, in both sexes. In ovariectomized rats, but not in controls, methadone nearly doubled the epoxide hydratase activity, whereas in male rats castration decreased the inductive effect of methadone. Gonadectomy had a significant effect on the results of methadone treatment with respect to glutathione S-transferase activity in female rats. A sex difference was noted in the control levels of aryl hydrocarbon hydroxylase and glutathione S-transferase, but not of epoxide hydratase activity. The glutathione S-transferase and aryl hydrocarbon hydroxylase activities were decreased in castrated male rats, whereas epoxide hydratase activity was unaltered. It is concluded that sex hormones play an important role in the induction of epoxide hydratase and glutathione S-transferase by methadone, but not of aryl hydrocarbon hydroxylase, at this particular dosage regime.     

Inhibition of purified glutathione S-transferases by indomethacin

Soluble rat liver glutathione S-transferases have been purified and a previously undescribed peak was observed. This peak contained glutathione S-transferase activity which was extensively inhibited by indomethacin. Glutathione conjugation of 1-chloro-2,4-dinitrobenzene by this isozyme, designated glutathione S-transferase VII, was inhibited 44 and 68% at indomethacin concentrations of 0.20 and 1.00 microM, respectively. The other six basic glutathione S-transferase isozymes were relatively unaffected by low concentrations of indomethacin. The pharmacological significance of this inhibition by indomethacin is largely dependent on the role of the glutathione S-transferase VII in leukotriene synthesis.     

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.     

Induction of translationally active rat liver glutathione S-transferase B messenger RNA by phenobarbital

Liver poly(A⁺)-RNA was isolated from untreated and phenobarbital-treated rats and translated in cell-free systems derived from wheat germ and rabbit reticulocyte lysates. The primary translation product of glutathione S-transferase B was comprised of two nonidentically sized subunits which comigrated on SDS-polyacrylamide gels with the purified glutathione S-transferase B subunits. The level of translatable glutathione S-transferase B mRNA in rat liver was elevated approximately 3 to 4-fold by phenobarbital administration. Our data suggest that chronic phenobarbital administration to rats increases the amount of cytosolic glutathione S-transferase B via an increase in the functional mRNA level encoding for the enzyme.     

The effect of dietary protein and sulfur amino acids on hepatic glutathione concentration and glutathione-dependent enzyme activities in the rat

Hepatic glutathione concentration and glutathione-dependent enzymes, glutathione S-transferase, glutathione peroxidase, and glutathione reductase, are important for protection against toxic compounds. Rats were fed diets containing 4, 7.5, 15, or 45% protein for 2 weeks. Glutathione and cysteine concentrations in rats fed the 4 and 7.5% protein diets were significantly lower (p less than 0.05) than in rats fed the 15 and 45% protein diets. Glutathione S-transferase activity increased with increasing dietary protein. Glutathione peroxidase activity was significantly lower (p less than 0.05) in rats fed 4 and 7.5% protein compared with rats fed 15 and 45% protein, whereas the activity of glutathione reductase was higher in rats fed 4 and 7.5% protein then in rats fed 15 or 45% protein. Dietary sulfur amino acids alone could account for the increase in glutathione concentration resulting from the increase in dietary protein from 7.5 to 15%. The limited availability of glutathione in animals fed the low protein diets could reduce the potential for detoxification of xenobiotics.     

Photoperiodic changes of the glutathione system of the brain under acute hypoxia]

The effect of acute hypoxic hypobaric hypoxia on the content of reduced glutathione and the activity of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione S-transferase, as well as 5'-nucleotidase in homogenates of juvenile male rats under conditions of varying photoperiodic duration: natural conditions of illumination, continuous illumination and continuous darkness were studied. Photoperiodic changes were revealed in the glutathione system of the control animals: the activity of glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase reduces under constant light, while the activity of glutathione peroxidase and glutathione S-transferase increases under conditions of constant darkness. The greatest inhibitory effect on the state of the glutathione system is brought about by constant light in case of acute hypoxia: the content of reduced glutathione decreases along with a sharp drop of the activity of glutathione S-transferase and glucose-6-phosphate dehydrogenase, observed against the background of decreased glutathione reductase activity. Permanent dark conditions eliminate partially or completely the negative effect of acute hypoxia on the glutathione system of the brain. The obtained results are indicator of a possibility of protecting role of melatonin in case of acute hypoxia.     

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).