Glutathione S-transferases of the bovine retina. Evidence that glutathione peroxidase activity is the result of glutathione S-transferase.

We have purified two isoenzymes of glutathione S-transferase from bovine retina to apparent homogeneity through a combination of gel-filtration chromatography, affinity chromatography and isoelectric focusing. The more anionic (pI = 6.34) and less anionic (pI = 6.87) isoenzymes were comparable with respect to kinetic and structural parameters. The Km for both substrates, reduced glutathione and 1-chloro-2,4-dinitrobenzene, bilirubin inhibition of glutathione conjugation to 1-chloro-2,4-dinitrobenzene, 1-chloro-2,4-dinitrobenzene inactivation of enzyme activity and molecular weight were similar. However, pH optimum and energy of activation were found to differ considerably. Retina was found to have no selenium-dependent glutathione peroxidase activity. The total glutathione peroxidase activity fractionated with the transferases in the gel-filtration range of mol.wt. 49000 and expressed activity with only organic hydroperoxides as substrate. Only the more anionic isoenzyme expressed both transferase and peroxidase activity.     

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.     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

Purification and properties of glutathione transferase from Issatchenkia orientalis.

Glutathione transferase (GST) (EC 2.5.1.18) was purified from a cell extract of Issatchenkia orientalis, and two GST isoenzymes were isolated. They had molecular weights of 37,500 and 40,000 and were designated GST Y-1 and GST Y-2, respectively. GST Y-1 and GST Y-2 gave single bands with molecular weights of 22,000 and 23,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GST Y-1 and GST Y-2 were immunologically distinguished from each other. GST Y-1 showed specific activity 10.4-times and 6.0-times higher when 1-chloro-2,4-dinitrobenzene and o-dinitrobenzene were used as substrates, respectively, than GST Y-2. GST activity was not detected for either isoenzyme when other substrates such as bromosulfophthalein and trans-4-phenyl-3-buten-2-one were used. GST Y-1 and GST Y-2 had Km values of 0.51 and 0.75 mM for glutathione, respectively, and of 0.16 and 4.01 mM for 1-chloro-2,4-dinitrobenzene. GST Y-1 was significantly inhibited by Cibacron blue 3G-A, and GST Y-2 was significantly inhibited by bromosulfophthalein.     

The glutathione S-transferase activity in the pyloric caeca of rainbow trout, Salmo gairdneri

Pyloric caeca of trout contain 1.9 mmol GSH/kg tissue. Cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.06 mmol/min/g protein. Cholate (3.3 mM) inhibits cytosolic transferase activity by 55% at pH 6.6 and by 4% at pH 7.4. The transferases do not bind 8-anilino-1-naphthalene sulphonate at pH 7.4. The cytosolic transferases are inactivated progressively by 1-chloro-2,4-dinitrobenzene, 50% of their activity being lost in 5.0 min. A minority of the activity does not bind to a glutathione-affinity matrix. At pH 6.6 its apparent Michaelis constants for GSH and 1-chloro-2,4-dinitrobenzene are 0.88 and 9.1 mM respectively. The rest of the activity is eluted from the affinity matrix as a single peak. Its apparent Michaelis constants for GSH and 1-chloro-2,4-dinitrobenzene are 0.33 and 2.9 mM respectively. Its subunit Mr is 22.4 kDa.     

Sex difference in subunit composition of hepatic glutathione S-transferase in rats

The activities of hepatic cytosolic glutathione S-transferases (GSTs) towards 1,2-dichloro-4-nitrobenzene in male rats were higher than those in females, however, the enzyme activities towards 1-chloro-2,4-dinitrobenzene were not significantly different between the two sexes. SDS-PAGE analysis of GSTs purified from male and female rat hepatic cytosols by affinity column chromatography showed that there was a significant difference in the subunit composition between the two sexes. With regard to the several isozymes of GSTs in male and female rats, isozymes with basic and neutral/acidic isoelectric points were separated into seven molecular species by chromatofocusing. These sex differences in the quantitative proportions of GST isozymes were also confirmed by immunotitration using anti-GST-BL and -AC antibodies. On the other hand, glutathione peroxidase (GSH-Px) activities in rat hepatic cytosol towards hydrogen peroxide and cumene hydroperoxide were markedly higher in females than in males. Of the two types of GSH-Px, selenoenzyme (Se-GSH-Px) and the Se-independent enzyme (non-Se-GSH-Px), the former was found to be mainly responsible for the sex difference in the enzyme activities. Moreover, the GSH-Px activity of GSTs, non-Se-GSH-Px, was also higher in females than that in males. Since GST isozymes of the BL type are known to possess GSH-Px activity towards cumene hydroperoxide, the increased activities of non-Se-GSH-Px in the female hepatic cytosol seemed to be mainly due to the increased transferase activities of the isozymes, GST-L2 and -BL.     

Purification and characterization of glutathione S-transferases from guinea pig liver

Four types of glutathione S-transferase were purified to homogeneity from guinea pig liver by DEAE-cellulose, Sephadex G-75, CM-cellulose, and affinity chromatography. These isozymes were named a, b, c, and d based on the reverse order of elution from a CM-cellulose column, and had specific activities of 89.6, 92.2, 99.0, and 44.0 units/mg, respectively, when assayed with 1 mM each of 1-chloro-2,4-dinitrobenzene and reduced glutathione. All four transferases of guinea pig liver were homodimers. The transferases b, c, and d had a similar molecular weight of 50,000 and their subunit sizes were 25,000, but the corresponding values for transferase a were 45,000 and 23,500, respectively. Transferase a was notably different in the activities towards organic hydroperoxides and 1,2-dichloro-4-nitrobenzene from the other isozymes. Transferases a and b, the major forms in guinea pig liver, were studied with respect to their biochemical properties, including kinetic parameters, absorption and fluorescence spectra, and bilirubin binding. Glutathione peroxidase activity of the transferase a was about 100 times higher than that of other isozymes. In guinea pig liver, it is estimated that transferase a is the major glutathione peroxidase, accounting for about 75% of the total organic hydroperoxide reduction.     

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.     

Purification and biochemical properties of glutathione S-transferase from Lactuca sativa

A glutathione S-transferase (GST) from Lactuca sativa was purified to electrophoretic homogeneity approximately 403-fold with a 9.6% activity yield by DEAE-Sephacel and glutathione (GSH)-Sepharose column chromatography. The molecular weight of the enzyme was determined to be approximately 23,000 by SDS-polyacrylamide gel electrophoresis and 48,000 by gel chromatography, indicating a homodimeric structure. The activity of the enzyme was significantly inhibited by ShexylGSH and S-(2,4-dinitrophenyl) glutathione. The enzyme displayed activity towards 1-chloro-2,4-dinitrobenzene, a general GST substrate and high activities towards ethacrynic acid. It also exhibited glutathione peroxidase activity toward cumene hydroperoxide.     

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.     

Isolation and characterization of an anionic glutathione S-transferase from rat liver cytosol

An anionic glutathione S-transferase representing approximately 20% of the total glutathione S-transferase protein and 10% of the total transferase activity toward 1-chloro 2,4-dinitrobenzene has been purified to homogeneity from the 105,000 x g supernatant of rat liver homogenate. The SDS gel electrophoretic data on subunit composition revealed that the anionic isozyme is composed of two subunits with an identical Mr of 26,000. The Km values for 1-chloro 2,4-dinitrobenzene and reduced glutathione were determined to be 0.94 mM and 0.23 mM respectively. A significant amount of glutathione peroxidase activity toward cumene hydroperoxide is associated with the new isozyme.     

Selective inhibition of glutathione S-transferases by 17 beta-estradiol disulfate.

Enzyme activity of homogeneous glutathione S-transferases A, B, and C with reduced glutathione and 1-chloro-2,4-dinitrobenzene was inhibited in varying degrees by 50 μm concentrations of monosulfate and disulfate derivatives of several steroids. In contrast, transferase AA activity was not affected. Of the inhibitors tested, estradiol-3,17-disulfate and estradiol-3-sulfate were the most inhibitory, followed by pregnenolone sulfate, estradiol-17-sulfate, dehydroisoandrosterone sulfate, and cortisol sulfate. Transferases A and C were most affected, especially by estradiol disulfate and estradiol-3-sulfate, which exhibited essentially complete inhibition at a concentration of μm. Double reciprocal plots of estradiol disulfate inhibition with respect to 1-chloro-2,4-dinitrobenzene concentration showed uncompetitive inhibition with transferases A and C and noncompetitive inhibition with transferase B (ligandin). With reduced glutathione as the variable substrate, transferases A and C exhibited noncompetitive inhibition kinetics, while transferase B showed partial noncompetitive kinetics.     

Rat glutathione transferase 8-8, an enzyme efficiently detoxifying 4-hydroxyalk-2-enals

Rat glutathione transferase 8-8 is one of the less abundant cytosolic glutathione transferases, accounting for approx. 1% of the total activity with 1-chloro-2,4-dinitrobenzene in liver. The enzyme is eluted at pH 6.3 upon chromatofocusing and has so far been identified in liver, kidney, lung and testis. Characteristic properties include high relative activity with ethacrynic acid (70% of the specific activity with 1-chloro-2,4-dinitrobenzene) and an apparent subunit Mr of 24 500. The most significant property noted is the high catalytic activity in the conjugation of 4-hydroxyalk-2-enals, major products of lipid peroxidation. The catalytic efficiency with these substrates exceeds corresponding values for all known substrates tested with any glutathione transferase, which suggests that transferase 8-8 may have evolved to detoxify 4-hydroxyalk-2-enals.     

Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree).

A hitherto unknown cytosolic glutathione S-transferase from rat liver was discovered and a method developed for its purification to apparent homogeneity. This enzyme had several properties that distinguished it from other glutathione S-transferases, and it was named glutathione S-transferase X. The purification procedure involved DEAE-cellulose chromatography, (NH4)2SO4 precipitation, affinity chromatography on Sepharose 4B to which glutathione was coupled and CM-cellulose chromatography, and allowed the isolation of glutathione S-transferases X, A, B and C in relatively large quantities suitable for the investigation of the toxicological role of these enzymes. Like glutathione S-transferase M, but unlike glutathione S-transferases AA, A, B, C, D and E, glutathione S-transferase X was retained on DEAE-cellulose. The end product, which was purified from rat liver 20 000 g supernatant about 50-fold, as determined with 1-chloro-2,4-dinitrobenzene as substrate and about 90-fold with the 1,2-dichloro-4-nitrobenzene as substrate, was judged to be homogeneous by several criteria, including sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, isoelectric focusing and immunoelectrophoresis. Results from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration indicated that transferase X was a dimer with Mr about 45 000 composed of subunits with Mr 23 500. The isoelectric point of glutathione S-transferase X was 6.9, which is different from those of most of the other glutathione S-transferases (AA, A, B and C). The amino acid composition of transferase X was similar to that of transferase C. Immunoelectrophoresis of glutathione S-transferases A, C and X and precipitation of various combinations of these antigens by antisera raised against glutathione S-transferase X or C revealed that the glutathione S-transferases A, C and X have different electrophoretic mobilities, and indicated that transferase X is immunologically similar to transferase C, less similar to transferase A and not cross-reactive to transferases B and E. In contrast with transferases B and AA, glutathione S-transferase X did not bind cholic acid, which, together with the determination of the Mr, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyse the reaction of menaphthyl sulphate with glutathione, and was in this respect dissimilar to glutathione S-transferase M; however, it conjugated 1,2-dichloro-4-nitrobenzene very rapidly, in contrast with transferases AA, B, D and E, which were nearly inactive towards that substrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

The glutathione transferase activity and tissue distribution of a cloned Mr28K protective antigen of Schistosoma mansoni.

A protective Mr28K antigen of Schistosoma mansoni, expressed from its cDNA, has been purified in a single step and shown to possess glutathione (GSH) transferase activity as predicted from sequence homologies with two mammalian GSH transferase multigene families. It is notable for its high 1-chloro-2,4-dinitrobenzene GSH transferase and linoleic acid hydroperoxide GSH peroxidase activities. The major GSH transferase of S. mansoni has been purified and its subunit is identical to this Mr28K antigen by criteria of Mr, immunochemistry, substrate specificity and peptide sequence analysis. In the parasite, the antigen is present in the tegument, protonephridial cells and subtegumental parenchymal cells. No significant immunological cross-reactivity between the S.mansoni and mammalian (human and rat) GSH transferases was observed.     

Purification and characterization of multiple glutathione S-transferase isozymes from Chironomidae larvae.

Glutathione S-transferase (GST) has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by Chironomidae larvae. We have purified and characterized GST from cytosolic fractions of Chironomidae larvae. GST with an M(r) of 23 kDa has been purified to homogeneity from larvae by centrifugation, size exclusion chromatography on Sephadex G25, and glutathione affinity and anion exchange chromatography. The purified enzyme exhibited moderate activity towards 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, 4-nitropyridine-N-oxide, p-nitrobenzyl chloride, ethacrynic acid, and cumene hydroperoxide. The enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 5.5. The enzyme had a maximum activity at approximately pH 8 and showed activity between 30 and 40 degrees C. It became inactive at higher temperature (>50 degrees C) for 5 min. The N-terminal sequence analysis of the amino acids shows a high % of conserved regions in the enzyme. The enzyme activity was comparable to levels of metabolism observed by animal GST involved in the detoxification of xenobiotics.     

Glutathione S-transferase composition of rat erythrocytes

With 1-chloro-2,4-dinitrobenzene as the electrophilic substrate, the specific activity of glutathione S-transferase in rat haemolysates was found to range from 0.002 to 0.013 mumol/min/mg haemoglobin at 30 degrees C. To establish the glutathione S-transferase composition, chromatofocusing was used which indicated the presence of a single soluble isoenzyme with an apparent pI of 6.1. A molecular weight of 48,000 was determined for the enzyme by gel filtration. The transferase enzyme in intact erythrocytes is shown to catalyze the formation of S-(2,4-dinitrophenyl)-glutathione from 1-chloro-2,4-dinitrobenzene and endogenous glutathione. Efflux of this conjugate from erythrocytes proceeded at a rate of 13 nmol/min/ml at 37 degrees C.     

Comparison of basal glutathione S-transferase activities and of the influence of phenobarbital, butylated hydroxy-anisole or 5,5'-diphenylhydantoin on enzyme activity in male rodents

1. Hepatic cytosolic glutathione S-transferase (GST) activities, toward five substrates, were shown to vary markedly among three laboratory rodent species. 2. Basal GST activities for 1-chloro-2,4-dinitrobenzene (hamster greater than mouse greater than rat), 1,2-dichloro-4-nitrobenzene (mouse greater than rat greater than hamster), p-nitrobenzyl chloride (rat = mouse = hamster), bromosulfophthalein (rat greater than mouse greater than hamster) and 1,2-epoxy-3-(p-nitrophenoxy)propane (mouse greater than rat = hamster) differed with respect to magnitude and distribution among species. 3. GST substrate activities in response to phenobarbital, butylated hydroxy-anisole or 5,5'-diphenylhydantoin treatment were increased more often in mouse and rat as compared to the hamster. 4. These results suggest that basal GST activity, as well as inducibility, differ among rodent species. Since GST are involved in detoxication processes, differences in GST properties may underlie variability in species sensitivity to toxicants.     

Glutathione S-transferase from beef heart: structural and kinetic properties

A simple and rapid method for the purification of glutathione S-transferase is described. The physical and kinetic properties of purified enzyme are reported. The protein is constituted of two identical subunits with a total molecular weight of 46,000 daltons. The isoelectric focusing of crude cytosol or purified preparation gives a single peak of activity with a pI of 7.1. The kinetic analysis shows a relatively strict substrate specificity. Only 1-chloro-2,4-dinitrobenzene is conjugated to reduced glutathione at an appreciable rate. The peroxidase activity of the enzyme with respect to cumene hydroperoxide as substrate is negligible. Hemin and bilirubin are competitive inhibitors of catalytic activity.     

The isolation and characterization of the major glutathione S-transferase from the squid Loligo vulgaris

1. The major glutathione S-transferase (GST) from the common squid Loligo vulgaris has been purified and shown to be a homodimer of subunit molecular mass 24,000 and pI 6.8. 2. It has high activity towards 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 4-hydroxynon-2-enal and linoleic acid hydroperoxide, low activity with 1,2-dichloro-4-nitrobenzene and no activity with ethacrynic acid, trans-4-phenyl-3-buten-2-one and 1,2-epoxy-3-(p-nitrophenoxy)propane. 3. The L. vulgaris GST did not cross-react with any of the available polyclonal antibodies raised against mammalian GSTs. 4. Forty amino acids of its N-terminal sequence have been determined. 5. Its activities and primary structure are compared with related proteins from other species.