Constitutive and Aroclor 1254-induced hepatic glutathione S-transferase, peroxidase and reductase activities in genetically inbred mice

1. Constitutive and Aroclor 1254-induced hepatic glutathione (GSH) S-transferases, GSH peroxidase and GSH reductase activities were determined in 12 strains of 8-10 week-old inbred male mice. 2. The constitutive GSH S-transferase activity varied from 2.5 (SJL/JCR) to 8.9 (C57BL/6N) mumol/min/mg protein and the corresponding values for the Aroclor 1254-treated mice were in the range of 7.1-23.0 mumol/min/mg protein. Aroclor 1254 significantly induced GSH S-transferase activity in all mice, however, significant interstrain differences were found in inducibility. 3. Aroclor 1254-treatment caused a 4.2-fold induction of GSH S-transferase in NFS/NCR but only a 1.4-fold increase in AKR/NCR mice. Aroclor 1254 significantly induced GSH reductase in all strains studied while GSH peroxidase activity decreased in these mice. 4. The range of hepatic GSH levels in control and Aroclor 1254-treated mice was relatively narrow for both groups (6.59-11.25 microM/g wet tissue).     

A rapid purification procedure for camel kidney glutathione S-transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37,000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 mumol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100. The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29,000 D and 26,000 D to give a native molecular weight of 55,000 D. The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. 1-chloro-2,4-dinitrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse. Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Distribution and some properties of the glutathione S-transferase and gamma-glutamyl transpeptidase activities of rainbow trout

1. Gills, kidney, intestinal caeca and liver of trout have glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene (200 500 nmol/min/mg protein), and reduced glutathione (0.5 2.0 mmol/kg tissue). 2. Only kidney and intestinal caeca have substantial gamma-glutamyl transpeptidase activity with gamma-glutamyl-rho-nitroanilide (2-9 nmol/min/mg protein). 3. Renal gamma-glutamyl transpeptidase is membrane-bound and has similar kinetic properties to its mammalian counterparts. 4. The data are consistent with the presence of a mercapturic acid pathway in trout.     

Induction of glutathione S-transferases in genetically inbred male mice by dietary ethoxyquin hydrochloride

1. Constitutive and ethoxyquin hydrochloride (EQ-HCl)-induced hepatic glutathione (GSH) S-transferase, GSH reductase, and GSH peroxidase activities were determined in 5 strains of 8-10 week old inbred male mice. 2. The constitutive GSH S-transferase (GST) activity varied from 2.9 (SJL/JCR) to 8.9 (C57BL/6NCR) mumol product formed/min/mg protein and the corresponding values for the EQ-HCl-treated mice were in the range of 15.3-25.3 mumol product formed/min/mg protein. 3. EQ-HCl induced GST activity in all the strains examined and this contrasted to the induction activity of Aroclor 1254 which was strain-dependent. GST activity was induced 2.9-fold in Aroclor 1254-responsive (C57BL/6) and 2.8-fold in non-responsive (DBA/2) mice, respectively.     

Effect of pineal indoles on activities of the antioxidant defense enzymes superoxide dismutase, catalase, and glutathione reductase, and levels of reduced and oxidized glutathione in rat tissues.

Male Sprague-Dawley rats were randomly divided into four groups. Two of the groups received a single intraperitoneal injection of melatonin and 5-methoxytryptamine (5 mg/kg body weight), respectively, at 9 PM. One group received an intraperitoneal injection of 5-methoxytryptophol (5 mg/kg body weight) at 9 AM. The remaining group received alcoholic saline (vehicle) and served as the control. All rats were sacrificed 90 min after injection and the livers, kidneys, and brains were dissected. The activities of superoxide dismutase, catalase, and glutathione reductase in the organs were measured. It was found that both melatonin and 5-methoxytryptamine were approximately equipotent in enhancing the activities of superoxide dismutase and glutathione reductase in the kidney and liver, while 5-methoxytryptophol displayed a weaker effect. Both melatonin and 5-methoxytryptamine augmented the level of reduced glutathione in the kidney and liver, while 5-methoxytryptophol did so only in the kidney. All three pineal indoles increased the activity of superoxide dismutase and lowered the ratio of oxidized to reduced glutathione in the brain.     

Isolation and characterization of the multiple glutathione S-transferases from human liver. Evidence for unique heme-binding sites

Thirteen forms of glutathione S-transferase were isolated from human liver in high yields by glutathione-affinity chromatography and chromatofocusing. Apparent isoelectric points ranged from 4.9 to 8.9 and included neutral forms. All 13 forms appeared to be identical immunochemically in a quantitative enzyme-linked immunosorbent assay. These forms were immunochemically distinct from the major acidic glutathione S-transferase found in placenta and erythrocyte and were immunochemically distinct from two forms of higher molecular weight glutathione S-transferase found in some but not all liver samples. The 13 forms exhibited similar activities with 1-chloro-2,4-dinitro-benzene as substrate, specific activities of 33-94 mumol/min/mg. Likewise, these forms all exhibited glutathione peroxidase activity with cumene hydroperoxide, specific activities of 1.5-8.3 mumol/min/mg. All 13 forms bound bilirubin with subsequent conformational changes leading to states devoid of transferase activity, a process prevented by the presence of foreign proteins. As hematin-binding proteins, however, these multiple transferases exhibited a very broad range of binding extending from nonbinding to high-affinity binding (KD approximately 10(-8) M). Hematin binding was noncompetitive with transferase activity and did not involve the bilirubin-binding site, suggesting the existence of unique heme-binding sites on these proteins. The two forms of the immunochemically distinct glutathione S-transferases transferases found in some liver samples also exhibited both transferase and peroxidase activities. In addition, they also have separate sites for binding bilirubin and hematin.     

Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase

A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.     

Organic nitrate reductase: reassessment of its subcellular localization and tissue distribution and its relationship to the glutathione transferases

1. Using a specific and sensitive GLC method for the determination of glyceryl trinitrate (GTN), its subcellular and tissue distribution were reassessed. Liver was the most active tissue, but activity was also detected in the heart, kidney and gut. In all tissues activity was localized in the soluble fraction. The activity of soluble glutathione S-transferase followed the same pattern, liver exhibiting the highest and the heart the lowest activity. 2. Pretreatment with phenobarbitone and 3-methylcholanthrene stimulated both the glutathione S-transferase and organic nitrate reductase activities. 3. Glutathione S-transferase activity was competitively inhibited by GTN. 4. A comparison of the plasma and hepatic metabolism of GTN revealed higher drug affinity for the hepatic enzyme.     

Plasma glutathione turnover in the rat: effect of fasting and buthionine sulfoximine

Hepatic glutathione (GSH) plays an important role in the detoxification of reactive molecular intermediates. Because of evidence that the intrahepatic turnover of glutathione in the rat may be largely accounted for by efflux from hepatocytes into the general circulation, the quantitation of plasma GSH turnover in vivo could provide a noninvasive index of hepatic glutathione metabolism. We developed a method to estimate plasma glutathione turnover and clearance in the intact, anesthetized rat using a 30-min unprimed, continuous infusion of 35S-labelled GSH. A steady state of free plasma glutathione specific radioactivity was achieved within 10 min, as determined by high-pressure liquid chromatography with fluorometric detection after precolumn derivatization of the plasma samples with monobromobimane. The method was tested after two treatments known to alter hepatic GSH metabolism: 90 min after intraperitoneal injection of 4 mmol/kg buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, and after a 48-h fast. Liver glutathione concentration (mean +/- SEM) was 5.00 +/- 0.53 mumol/g wet weight in control rats. It decreased to 3.10 +/- 0.35 mumol/g wet weight after BSO injection and to 3.36 +/- 0.14 mumol/g wet weight after fasting (both p less than 0.05). Plasma glutathione turnover was 63.0 +/- 7.46 nmol.min-1.100 g-1 body weight in control rats, 35.0 +/- 2.92 nmol.min-1.g-1 body weight in BSO-treated rats, and 41.7 +/- 2.28 nmol.min-1.g-1 body weight after fasting (both p less than 0.05), thus reflecting the hepatic alterations. This approach might prove useful in the noninvasive assessment of liver glutathione status.     

Multiple forms of glutathione S-transferase from pig liver--reaction with methyl linoleate hydroperoxides

1. Seven isoenzyme forms of glutathione S-transferase were purified from pig liver. 2. The most basic isoenzyme reduced methyl 13-hydroperoxy-cis-9,trans-11-octadecadienoate in the absence of detergent at a higher rate (0.3 mumol/min/mg protein) than predicted from substrate solubility. 3. This demonstrates that glutathione transferase possesses some surface acting character for neutral lipid hydroperoxides.     

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.     

Tissue-specific expression of the rat glutathione S-transferases

Tissue-specific patterns of rat glutathione S-transferase expression have been demonstrated by in vitro translation of purified poly(A) RNAs and by protein purification. Poly(A) RNAs from six rat tissues including heart, kidney, liver, lung, spleen, and testis were used to program in vitro translation with the rabbit reticulocyte lysate system and [35S]methionine. The glutathione S-transferase subunits synthesized in vitro were purified from the translation products by affinity chromatography on S-hexylglutathione-linked Sepharose 6B columns. The affinity bound fractions were analyzed by Na dodecyl SO4-polyacrylamide gel electrophoresis and fluorography. A subunit of Mr = 22,000 detected in the in vitro translation products of poly(A) RNAs from heart, kidney, lung, spleen, and testis is missing from the translation products of liver poly(A) RNAs. This Mr = 22,000 subunit is present only in the anionic glutathione S-transferase fraction purified from rat heart, kidney, lung, spleen, and testis. Purified anionic glutathione S-transferase from rat liver does not contain this subunit. The relative specific activities toward a dozen different substrates also demonstrate the nonidentity between liver and kidney anionic glutathione S-transferases. In addition, among the glutathione S-transferase subunits expressed in the liver, some of them could not be detected in the other tissues investigated. Our results indicate that tissue-specific expression of rat glutathione S-transferases may occur pretranslationally.     

Purification and some properties of glutathione S-transferase from Escherichia coli B.

Glutathione S-transferase was purified approximately 2,300-fold from cell extracts of Escherichia coli B with a 7.5% activity yield. The molecular weight of the enzyme was 45,000, and the enzyme appeared to consist of two homogeneous subunits. The enzyme was almost specific to 1-chloro-2,4-dinitrobenzene (Km, 1.43 mM) and glutathione (Km, 0.33 mM). The optimal pH and optimal temperature for activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable from pH 5 to 11. The activity of the enzyme for 1-chloro-2,4-dinitrobenzene (3,2 mumol/min per mg of protein) was significantly lower than those of the enzymes from mammals, plants, and fungi.     

Oral glutathione increases tissue glutathione in vivo

Mice were given an oral dose of glutathione (GSH) (100 mg/kg) and concentrations of GSH were measured at 30, 45 and 60 min in blood plasma and after 1 h in liver, kidney, heart, lung, brain, small intestine and skin. In control mice, GSH concentrations in plasma increased from 30 microM to 75 microM within 30 min of oral GSH administration, consistent with a rapid flux of GSH from the intestinal lumen to plasma. Under these GSH-sufficient conditions, no increases over control values were obtained in GSH concentrations in most tissues except lung over the same time course. Mice pretreated for 5 days with the GSH synthesis inhibitor, L-buthionine-S,R-sulfoximine (BSO, 80 mumol/day) had substantially decreased tissue concentrations of GSH. Oral administration of GSH to these GSH-deficient animals gave statistically significant increases in GSH concentrations in kidney, heart, lung, brain, small intestine and skin but not in the liver. Administration of the equivalent amount of the constituent amino acids, glutamate, cysteine, and glycine, resulted in little change in GSH concentrations in all tissues in GSH-deficient animals. Thus, the results show that oral GSH can increase GSH concentrations in several tissues following GSH depletion, such as can occur in toxicological and pathological conditions in which GSH homeostasis is compromised.     

Modulation of selenium-dependent glutathione peroxidase (Se-GSH-Px) activity in mice

Selenium-dependent glutathione peroxidase activity was assessed in the liver, kidney, lung and blood of mice from seven strains (129/ReJ, BALB/c, C3H/HeSnJ, C3H/S, C57BL/6J, Csb, and S.W.) at five ages (newborn, 21, 70, 175 and +500 days old). Activity was highest in the liver (0.25 U/mg protein) followed by blood hemolysate (0.16 U/mg protein) with kidney and lung displaying similar, comparatively lower levels of activity (0.14 and 0.12 U/mg protein respectively). Although activity was shown by statistical analysis to be not significantly different among the strains (p = 0.05), age-associated, strain-specific changes in enzyme activity were noted to be highly significant (p = 0.001). Also, ethanol administered in drinking water resulted in a marked reduction in selenium-dependent glutathione peroxidase activity during both short- (1-2 weeks) and long- (5-6 weeks) term treatment periods. Changes in this enzyme due to aging and after exposure to xenobiotics such as ethanol may have serious ramifications given the importance of this enzyme in the detoxification of reactive oxygen metabolites.     

东亚飞蝗谷胱甘肽S-转移酶分离纯化

通过硫酸铵沉淀技术和GSH-agarose亲和层析对东亚飞蝗Locusta migratoria manilensis(Meyen)5龄若虫谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)进行了分离纯化。结果表明GSTs活性在硫酸铵各沉淀段均有分布,但在55%～100%沉淀段活性较高,在硫酸铵饱和度为85%时比活力最高,达到420.33μmol/min/mg protein,纯化倍数为18.86。根据硫酸铵粗沉淀谷胱甘肽S-转移酶结果,选择硫酸铵浓度为60%～90%沉淀段进行GSH-agarose亲和层析,纯化后比活力最高达到1365.29μmol/min/mg protein,纯化倍数达到61.25。经SDS-PAGE鉴定,得到的GST为1条带,亚基的分子量约为24kDa。     

Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties

Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.     

Organ distribution of aldehyde dehydrogenase activity in the rainbow trout (Salmo gairdneri Richardson)

1. Aldehyde dehydrogenase activity was measured in gills, muscle, brain, intestine, kidney, heart and liver of rainbow trout, using 3,4-dihydroxyphenylacetaldehyde (the biogenic aldehyde derived from dopamine) as the substrate. 2. Aldehyde dehydrogenase activity was found to be present in all of the organs studied. 3. The highest activity was found in the liver (276 nmol/min.g wet wt of tissue). 4. A remarkably high activity was found in the heart (117 nmol/min.g). 5. The gills showed the lowest activity (1.9 nmol/min.g).     

Effects of paraquat on anti-oxidant system in rats

The toxic effects of paraquat on the anti-oxidant defense system of male albino rats were evaluated, after administering either a single dose (1.5 and 7.5 mg/kg of body weight) or continuous daily doses (same as above, i.e., 1.5 mg/kg and 7.5 mg/kg of body weight) for 3 and 7 days. Glutathione levels in blood cells, liver, lung and kidney tissues decreased in a dose and time dependent manner. Glutathione reductase and glucose-6-phosphate dehydrogenase activity decreased, whereas the activity of glutathione-S-transferase, glutathione peroxidase, catalase and superoxide dismutase increased in paraquat exposure. Malondialdehyde formation also increased in a dose and time dependent manner. The alterations of anti-oxidant system particularly glutathione can be utilized as biomarkers during management of paraquat poisoning.     

Modulatory influence of Brassica compestris Linn var sarson on phase-II carcinogen metabolizing enzymes and glutathione levels in mice

The present study reports the modulatory influence of 95% ethanolic extract from the seeds of B. compestris on the activity of phase-II enzymes such as glutathione S-transferase (GST), DT-diaphorase (DTD) and reduced glutathione (GSH) level in the skin, lung, kidney and forestomach of the mouse. Oral treatment with the seed extract at 800 mg/kg body wt. for 15 days significantly elevated GST in lung and forestomach and DT-diaphorase in forestomach and skin and GSH level in lung, kidney forestomach and skin. The lower dose 400 mg/kg body wt was effective only in inducing GST and DT-diaphorase activity in forestomach and reduced glutathione level in lung. The findings suggest that B. compestris seed extract may block or suppress the events associated with chemical carcinogenesis at least in part, by inducing metabolic detoxification of the carcinogen.