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.     

Interrelationship between cationic and anionic forms of glutathione S-transferases of bovine ocular lens.

Since the eye is constantly exposed to potentially damaging chemical compounds present in the atmosphere and vascular system, we investigated the physiological role of glutathione S-transferase (GSH S-transferase) in detoxification mechanisms operative in the ocular lens. We have purified an anionic and a cationic GSH S-transferase from the bovine lens to homogeneity through a combination of gel filtration, ion-exchange and affinity chromatography. The anionic (pI 5.6) and cationic (pI 7.4) S-transferases were found to have distinct kinetic parameters (apparent Km and Vmax. pH optimum and energy of activation). However, both species were demonstrated to have similar molecular weights and amino acid compositions. Double-immunodiffusion and immunotitration studies showed that both lens S-transferases were immunologically similar. The very close similarity in amino acid compositions and immunological properties strongly indicates that these two transferases either originate from the same gene or at least share common antigenic determinants and originate from similar genes. The bovine lens GSH S-transferases had no glutathione peroxidase activity with either t-butyl hydroperoxide or cumene hydroperoxide as substrate. However, the antibody raised against the homogeneous anionic glutathione S-transferase from the bovine lens was found to precipitate both glutathione S-transferase and glutathione peroxidase activities out of solution in the supernatant of a crude bovine liver homogenate.     

The hepatic glutathione transferases of the male little skate, Raja erinacea

Five cytosolic glutathione transferases were isolated from the liver of the male little skate, Raja erinacea, a marine elasmobranch. They were designated E-1 through E-5 in order of their elution from a DEAE-cellulose column with a 0 to 100 mM KCl gradient in 0.01 M Tris (pH 8.0). Each eluted peak of glutathione transferase activity, after concentration, was applied to an affinity column prepared by reaction of epoxy-activated Sepharose 6B with glutathione (GSH). Elution of the various glutathione transferases from this column with GSH resulted in the further purification of each enzyme; the major glutathione transferase, E-4 and E-1, were purified to apparent homogeneity by this procedure. Skate glutathione transferase E-4 is dimeric and the subunits are either very similar or identical in molecular weight (about 26 000 daltons). Enzymes E-2 through E-5 were acidic proteins (pI less than 7.0) and had high specific glutathione transferase activity (0.3--12 mumol/min/mg protein) with benzo[a]pyrene 4,5-oxide (BPO) as substrate, whereas the other enzyme (E-1) had low activity (0.01 mumol/min/mg) with BPO and a basic pI (greater than 9.5). Bilirubin and hematin, non-substrate ligands, bound tightly to homogeneous E-4, with dissociation constants in the micromolar range.     

Separation of glutathione transferase subunits from Proteus vulgaris by two-dimensional gel electrophoresis

Cytosolic glutathione transferases of Proteus vulgaris were purified by affinity chromatography and characterized by two-dimensional gel electrophoresis. Four different subunits were identified, and each subunit contained a different molecular mass, ranging from 26.2 kDa to 28.5 kDa; a different pI value, ranging from 8.2 to 9.4; and a different amount of protein fraction, ranging from 10% to 56%. All four subunits existed as basic proteins (pI > 7.0). From these results, we concluded that multiple forms of glutathione transferase enzymes existed in Proteus vulgaris, and four different glutathione transferase subunits were separated by 2-D gel electrophoresis.     

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.     

Two immunologically distinct Yc-type subunits are present in rat lung glutathione S-transferases.

Two types of 25 000-Mr subunits are present in rat lung glutathione S-transferase I (pI 8.8). These subunits, designated Yc and Yc', are immunologically and functionally distinct from each other. The homodimers YcYc (pI 10.4) and Yc'Yc' (pI 7.6) obtained by hybridization in vitro of the two subunits of glutathione S-transferase I (pI 8.8) were isolated and characterized. Results of these studies indicate that only the Yc subunits express glutathione peroxidase activity and cross-react with the antibodies raised against glutathione S-transferase B (YaYc) or rat liver. The Yc' subunits do not express glutathione peroxidase activity and do not cross-react with the antibodies raised against glutathione S-transferase B of rat liver. The amino acid compositions of these two subunits are also different. These two subunits can also be separated by the two-dimensional gel electrophoresis of glutathione S-transferase I (pI 8.8) of rat lung.     

Purification and characterization of glutathione transferase of human thyroid

An anionic (pI 4.6) isoenzyme of glutathione transferase was purified to homogeneity from human thyroid by affinity chromatography followed by isoelectric focusing. The content of enzyme was calculated to constitute about 0.2% of soluble proteins. The enzyme is formed by two identical subunits of 23,000 daltons approximately. The thyroid transferase did not catalyze the reduction of peroxides. Physical, catalytic and immunological analyses demonstrated extensive similarities between the thyroid transferase and the transferase from placenta, erythrocytes and breast. On the other hand, the thyroid transferase appears catalytically different from transferase 7-7, even if both cross-react with the antibodies raised against human placenta transferase.     

Purification and characterization of a new form of glutathione S-transferase from human erythrocytes

Presence of a new form of glutathione S-transferase has been demonstrated in human erythrocytes. using two different affinity ligands this enzyme has been separated from the previously characterized glutathione S-transferases ?. The new enzyme is highly basic with a pI of > 10. The new enzyme which represents less than 5 percent of glutathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene as substrate and about 10 percent of total glutathione S-transferase protein of erythrocytes has different amino acid composition, substrate specificities, and immunological characteristics from those of the major erythrocyte glutathione S-transferase ?. Immunological properties of the new enzyme indicate that this form may be different from other glutathione S-transferases of human tissues.     

Effect of anaerobic environment on the glutathione transferase isoenzymatic pattern in Proteus mirabilis

When Proteus mirabilis was cultured anaerobically in the presence of nitrate as terminal electron acceptor, a dramatic reduction of glutathione transferase production occurred. The analysis of the glutathione affinity purified materials in terms of substrate specificity, SDS-PAGE pattern, IEF pattern and immunoblotting revealed that a significantly different glutathione transferase pattern also occurred: two new glutathione transferase forms with an isoelectric point at pH 4.8 and 5.0 appeared. Their N-terminal amino acid sequence analysis as well as the ability to bind to a glutathione affinity column indicate that major differences between anaerobic and aerobic glutathione transferase forms are mainly located in the C-terminal region of the primary structure. In contrast, no significant changes occurred in the production of glutathione transferase isoenzymes when P. mirabilis was grown anaerobically in the absence of a terminal electron acceptor. These results support the idea that bacterial glutathione transferase expression is not strictly related to the absence of oxygen stress.     

Identification of a novel glutathione transferase in human skin homologous with class alpha glutathione transferase 2-2 in the rat.

Six forms of glutathione transferase with pI values of 4.6, 5.9, 6.8, 7.1, 8.5 and 9.9 have been isolated from the cytosol fraction of normal skin from three human subjects. The three most abundant enzymes were an acidic Class Pi transferase (pI 4.6; apparent subunit Mr 23,000), a basic Class Alpha transferase (pI 8.5; apparent subunit Mr 24,000) and an even more basic glutathione transferase of Class Alpha (pI 9.9; apparent subunit Mr 26,500). The last enzyme, which was previously unknown, accounts for 10-20% of the glutathione transferase in human skin. The novel transferase showed greater similarities with rat glutathione transferase 2-2, another Class Alpha enzyme, than with any other known transferase irrespective of species. The most striking similarities included reactions with antibodies, amino acid compositions and identical N-terminal amino acid sequences (16 residues). The close relationship between the human most basic and the rat glutathione transferase 2-2 supports the classification of the transferases previously proposed and indicates that the similarities between enzymes isolated from different species are more extensive than had been assumed previously.     

Heterogeneity of dog-liver glutathione S-transferases. Evidence for a unique temperature dependence of the catalytic process

Dog liver glutathione S-transferase activities are associated with five cytosolic proteins and to approximately 1.5% with microsomal proteins determined on the basis of activity conjugating to 1-chloro-2,4-dinitrobenzene. The four major cytosolic enzymes were purified to apparent homogeneity by sequential use of ion-exchange, hydrophobic, hydroxyapatite and affinity chromatography. The isolated transferases are binary combinations of three classes of subunits: alpha (Mr = 26,000), beta (Mr = 27,000), gamma (Mr = 28,500). They were classified by roman numerals assigned in order of increasing isoelectric point as DI alpha gamma (pI 6.4), DII alpha alpha (pI 6.9), DIII beta gamma (pI 8.1), and DIV beta gamma (pI 8.7). Additionally, traces of conjugating activity may be attributed to a, beta monomeric or dimeric protein with cationic character. The differences in catalytic specificity, temperature and pH dependence of activity, and sensitivity and kinetic response to inhibitory ligands may reflect the intrinsic structural heterogeneity of the transferases. At physiological glutathione concentrations DI alpha gamma accounted for roughly 60% of the total 1-chloro-2,4-dinitrobenzene-conjugating activity, the rank order of activity being DI alpha gamma greater than DII alpha alpha greater than DIV beta gamma greater than DIII beta gamma. The glutathione-dependent denitration of organic nitrates seems to be restricted to the cationic enzymes, whereas 1,2-dichloro-4-nitrobenzene-conjugating activity is exclusively associated with the anionic transferases, DI alpha gamma much greater than DII alpha alpha. Arrhenius plots from initial rate experiments performed over a range of temperatures (15-40 degrees C) exhibit an upward bend for DI alpha gamma, an apparently constant slope for DII alpha alpha and DIII beta gamma, and a downward bend for DIV beta gamma.     

Purification and physicochemical characterization of a recombinant phospholipid hydroperoxide glutathione peroxidase from Oryza sativa

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. In the present work, the entire encoding region for Oryza sativa PHGPx was expressed in Escherichia coli M15, and the purified fusion protein showed a single band with 21.0 kD and pI = 8.5 on SDS- and IFE-PAGE, respectively. Judging from CD and fluorescence spectroscopy, this protein is considered to have a well-ordered structure with 12.2% alpha-helix, 30.7% beta-sheet, 18.5% gamma-turn, and 38.5% random coil. The optimum pH and temperature of the enzyme activity were pH 9.3 and 27 degrees C. The enzyme exhibited the highest affinity and catalytical efficiency to phospholipid hydroperoxide employing GSH or Trx as electron donor. Moreover, the protein displayed higher GSH-dependent activity towards t-Butyl-OOH and H(2)O(2). These results show that OsPHGPx is an enzyme with broad specificity for hydroperoxide substrates and yielded significant insight into the physicochemical properties and the dynamics of OsPHGPx.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.     

Superoxide dismutase, glutathione peroxidase, and glutathione reductase in sheep organs

The enzyme activities of the superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione reductase (GR) and thiobarbituric acid reactive substances (TBARS) content were measured in tissue extracts of the liver, kidney and lung of sheep in a nonpolluted control area (C), a polluted area pasture (PP) and those from polluted areas but fed in the laboratory with an experimental emission supplement diet (EEF). Compared with the control SOD, activity was significantly increased (1.75 times) only in the liver of the PP group. In the EEF group there was a tendency toward lower activities in all organs. The Cu,Zn-SOD isoenzymes pattern analyzed by isoelectrofocusing was different in the organs of the animals exposed to pollutants when compared with those of the controls. In the liver, two new isoenzymes with pI 5.30 and 5.70 were found in the PP group and an additional isoenzyme with pI 5.10 in the EEF group. The kidney isoenzymes with pl 5.30 and 5.40 were inhibited in the EEF group. In the lung, two new isoenzymes appeared with pl 5.30 and 5.40 in the PP group and two new isoenzymes with pI 6.10 and 6.50 in the EEF group. GSHPx activity was inhibited in the liver and kidney of the sheep exposed to pollutants. GR activity was significantly changed only in the liver. The activity in the PP group was 2.30 and 2.10 times higher than in the C and EEF groups, respectively. TBARS content was increased in the liver and kidney of the EEF group compared with the control.     

Occurrence of two isoforms of glutathione reductase in the green alga Chlamydomonas reinhardtii

Two isoforms (isoenzymes) of glutathione reductase (NADPH: oxidized glutathione oxidoreductase, EC 1.6.4.2; GR) were clearly resolved when enzyme preparations partially purified from the unicellular alga Chlamydomonas reinhardtii were subjected to column chromatofocusing in the pH range from 8 to 4. One isoform (GR I) exhibited an almost electroneutral isoelectric point (pI, 6.9–7.1) and the other (GR II) was a very acidic protein (pI, 4.7–4.9). Both GRs are, however, homodimeric flavoproteins with similar molecular masses of approx. 127 kDa. Cross-reaction with an antibody against the cyanobacterial GR allowed determination of their subunit molecular masses by Western blotting after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a value of 66 kDa being estimated in both cases. The two algal GR isoforms showed similar K _m values for the oxidized form of glutathione (approx. 50 M). However, the K _m values for NADPH were different, being 7 M and 28 M for GR I and GR II, respectively. The two isoforms also differed in their optimum pH. Thus, whereas GR I showed a clear maximum at neutral pH, GR II exhibited a broader optimum around pH 8.5 and was more active in the alkaline range. The relative contribution of the two isoforms to the total activity in enzyme preparations of cells disrupted by two different methods indicates that GR I should be a cytoplasmic isoform and GR II a plastidic isoform. The physiological roles of the GR isoenzymes found in Chlamydomonas are discussed and some of their properties compared with those of GRs isolated from other photosynthetic organisms.Abbreviations GSSG glutathione, oxidized form - GR NAD-PH-glutathione reductase (EC 1.6.4.2) - G3P glyceraldehyde-3-phosphate - pI isoelectric point - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate This work was supported in part by grants NO. PB 87–401, PB 90–99 and BIO 91–1078 of the DGICYT (Ministerio de Educatión y Ciencia, Spain) and the Autonomous Government of Andalusia (Spain). Postdoctoral aid from the Alexander von Humboldt Foundation (Bonn, FRG) to A.S. is also acknowledged.     

Interaction of 1,4-benzoquinone and 2,4-dichlorophenoxyacetic acid with microsomal glutathione transferase from rat liver

Glutathione transferase (GST) was purified from the microsomes of rat liver by glutathione affinity chromatography. The interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1,4-benzoquinone with microsomal GST was investigated and compared with cytosolic GST. The kinetic inhibition pattern of 1,4-benzoquinone towards microsomal GST was found to be different from that towards cytosolic GST. Microsomal GST purified by affinity chromatography was inhibited by 2,4-D in a non dose-dependent manner, while the crude microsomal GST was inhibited in a dose-dependent manner. This difference was shown to be induced by a reaction on the affinity column, and not by Triton X-100 (also shown to be a GST inhibitor), glutathione, or the elution buffer 0.2% Triton X-100 and 5 mM glutathione in 50 mM Tris-HCl, pH 9.6. The binding of microsomal GST to the affinity matrix caused a partial inactivation of the active site for 2,4-D interaction. The results show that the properties of soluble GST enzymes may not be extrapolated to the microsomal ones.     

Newly identified bile acid binders in rat liver cytosol. Purification and comparison with glutathione S-transferases

Gel filtration of male rat liver cytosol preincubated with radiolabeled lithocholic, chenodeoxycholic, and glycochenodeoxycholic acids, and taurocholic acid revealed two major peaks of radioactivity, one co-eluting with the glutathione S-transferases and the other with a separate fraction, respectively. Chromatofocusing of the pooled fractions containing the new bile acid binding activity resulted in a separation of bile acid binding from the previously described organic anion binding activity in this fraction. Two binding peaks for lithocholic acid (pI 5.6, Binder I, and pI 5.5, Binder II) were identified on chromatofocusing and were further purified to apparent homogeneity by hydroxyapatite chromatography. The two Binders were monomers having identical molecular weight (33,000) and similar amino acid compositions. Bile acid binding to purified Binders I and II and glutathione S-transferases A, B, and C was studied by inhibition of the fluorescence of bound 1-anilino-8-naphthalenesulfonate (ANS). Confirmatory experiments using equilibrium dialysis produced comparable results. Glutathione S-transferase B had greater affinity for bile acids than transferases A or C. Binder II, which had greater affinity than Binder I for most bile acids, had greater affinity for chenodeoxycholic acid than transferase B but comparable or lower affinities for the other bile acids. All bile acids studied diminished ANS fluorescence with Binder II. Taurocholic and cholic acids increased ANS fluorescence with Binder I without affecting KANS, whereas lithocholic and chenodeoxycholic acids diminished ANS fluorescence with Binder I. In summary, we have identified and isolated two proteins (Binders I and II) which, along with glutathione S-transferase B, are the major hepatic cytosol bile acid binding proteins; these proteins have overlapping but distinct specificities for various bile acids.     

Purification and characterization of camel liver glutathione S-transferase

• 1.1. Glutathione S-transferases have been purified (18-fold) in 65–70% yield from the liver of one humped camel using affinity chromatography on glutathione-linked agarose.
• 2.2. Chromatofocusing technique resolves the glutathione S-transferases into seven distinct isoenzymes with apparent pI of 8.7, 8.4, 8.0, 7.8, 7.3 and 6.5.
• 3.3. The major isoenzyme (pI 8.7) which accounted for over 95% of the total activity was composed of two identical subunits of molecular mass 24,000 and was immunologically similar to the other six isoenzymes.
• 4.4. The substrate specificities and the effect of various inhibitors on the activity of the abundant camel liver isoenzyme were also examined.

     

Inhibition of glutathione reductase by oncomodulin

Evidence for a specific interaction between oncomodulin and glutathione reductase is presented. Glutathione reductase (EC 1.6.4.2) isolated from either the bovine intestinal mucosa or the rat liver was bound in a Ca2(+)-dependent manner to oncomodulin which was covalently attached to Sepharose. In addition, glutathione reductase was able to catalyze the reduction of the disulfide-linked dimer of oncomodulin. The interaction of these proteins could also be indirectly demonstrated by monitoring glutathione reductase activity since oncomodulin was shown to inhibit the enzyme in a dose-dependent manner with an apparent IC50 of approximately 5 microM. The kinetic analysis of the oncomodulin-dependent effects on glutathione reductase activity indicates that oncomodulin interacts at a site other than the active site as the oncomodulin-induced inhibition was of the noncompetitive type. The in vivo inhibition of glutathione reductase appears to be an oncomodulin-specific effect as closely related members of the troponin C superfamily such as rabbit (pI 5.5) or carp (pI 4.25) parvalbumins, as well as calmodulin, failed to affect the activity of this enzyme. The present in vitro study indicating that oncomodulin can regulate the activity of glutathione reductase could be very significant with respect to the elucidation of a physiological role for oncomodulin.     

Purification and characterization of glutathione S-transferase isozymes in dog lens.

1. Two isozymes of glutathione S-transferase (GST-dl1 and GST-dl2) were purified to homogeneity from dog lens. 2. The subunit size and the isoelectric point were determined to be 24,000 and > pI 9.5 for GST-dl1 and 22,000 and pI 8.1 for GST-dl2. 3. It was judged that GST-dl1 is a class alpha enzyme and GST-dl2 belongs to class pi on the basis of their immunological properties and N-terminal amino acid sequences. 4. The expression pattern of glutathione S-transferase isoenzymes in dog lens is different from that in pig, rat and bovine lenses.