Purification Of Glutathione S-transferase Research Articles

PLEF, a novel vector for expression of glutathione S-transferase fusion proteins in mammalian cells

An expression vector, pLEF, has been used to produce the intracellular domain (IC) of the human CD95 (Fas/APO-1) apoptosis receptor as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts. GST::CD95IC was affinity-purified in a single step using glutathione-Sepharose. Purification of GST::CD95IC from 32P-labelled L929 cells and cleavage with thrombin revealed that CD95IC was phosphorylated in vivo when produced as a GST fusion protein. Therefore, pLEF may facilitate the mapping of in vivo-modified sites of eukaryotic proteins

Purification of Glutathione S-Transferases from Yarrowia lipolytica

Purification of Glutathione S-Transferases from Yarrowia lipolytica

Conjugation of microsome generated and synthetic aflatoxin B 1-8,9 epoxide and styrene oxide to glutathione by purified glutathione S-transferases from hamster and mouse livers

Glutathione (GSH) conjugation of microsome-mediated and synthetic aflatoxin B 1 (AFB 1)-epoxide and styrene oxide has been studied with purified glutathione transferases (GSTs) from mouse and hamster liver cytosols. In hamster, with microsomally activated epoxide, the alpha group of GSTs show about 10-fold more activity than the mu group. With the synthetic AFB 1 epoxide, the mu enzymes designated H 3B and C show considerable activity although less than alpha, whereas H 3A and D demonstrate similar ranges of activity as the alpha group. The pi class of GST could not be assayed due to its absence in the hamster liver. The mouse liver cytosols show 3.6-fold greater activity than hamster cytosol in microsome mediated assay system. The mouse alpha and mu enzymes have similar levels of activity in the microsome mediated system; this activity could not be determined with the pi GST due to shortage of this enzyme. The alpha group has 2- and 5-fold higher activity than mu and pi group of GSTs, respectively, with the synthetic epoxide of AFB 1. With styrene oxide, the purified GSTs from hamster liver show total loss of activity whereas in the mouse alpha, mu and pi classes of GSTs have similar range of activity as the cytosol. The role of alpha and mu isozymes of GST in rendering these animals resistant to hepatocarcinogenecity is suggested.

Enantiotopic Selectivity in Monodemethylation of Fenitrothion by Rat Liver Glutathione <i>S</i>-Transferase Isoenzymes and Mouse Liver Cytosol

Monodemethylation of fenitrothion catalyzed by any of five purified glutathione S-transferases from rat liver predominantly produced the (S P ) -(-)-enantiomer of desmethyl fenitrothion. Mouse liver cytosolic fraction demethylated fenitrothion in the presence of glutathione to produce predominantly the (R) P -(+)-isomer. The absolute configuration of the (+)-desmethyl fenitrothion was determined as (R) P by X-ray crystallography of its brucine salt

Glutathione-dependent biotransformation of methyl parathion by mouse liver in vitro

The present study was undertaken in an attempt to reconcile the seemingly conflicting observations that glutathione-dependent biotransformation of methyl parathion ( O,O-dimethyl- O-(4-nitrophenyl) phosphorothionate) by hepatic supernatant or partially purified glutathione S-transferases occurs in vitro, but not to any significant degree in vivo in the mouse. While incubation of 20 μM methyl parathion with glutathione-fortified 100 000 × g hepatic supernatant resulted in biotransformation of this insecticide, addition of the carbon monoxide exposed microsomal fraction (without NADPH) to the supernatant abolished this metabolism. HPLC analyses of the distribution of methyl parathion between 100 000 × g supernatant and carbon monoxide-exposed microsomes revealed that little methyl parathion could be recovered in the 100 000 × g supernatant, and that the bulk of this insecticide was associated with the microsomal fraction. Increasing the concentration of methyl parathion to 1 mM resulted in a greater fraction of methyl parathion found in the supernatant compared to that with 20 μM, although the bulk of methyl parathion remained associated with the microsomal fraction. While this increase in the fraction of substrate located within the supernatant led to limited glutathione-dependent metabolism of methyl parathion, it must be emphasized that a liver concentration of 1 mM methyl parathion is far greater than that which could be achieved in vivo. In conclusion, the results of the present study support the hypothesis that in the mouse, glutathione- dependent metabolism of methyl parathion does not occur to a significant degree in vivo because of its limited access to the soluble glutathione S-transferases.

Glutathione conjugation of microsome-mediated and synthetic aflatoxin B 1-8,9-oxide by purified glutathione S-transferases from rats

Glutathione (GSH) conjugation of microsome-mediated and synthetic aflatoxin B 1 (AFB 1)-epoxide and styrene oxide has been investigated with purified GSH S-transferases (GSTs) from rats. Both styrene oxide and AFB 1-epoxide were conjugated preferentially by mμ GSTs 3-3, 3–4 and 4-4 as compared to alpha GSTs 1-1, 1–2 and 2-2. The highest catalytic activity with styrene oxide conjugation was associated with GST 4-4. The highest catalytic activity with microsome-mediated AFB 1-epoxide conjugation was observed with GST 3-3 whereas with the synthetic AFB 1-epoxide conjugation was seen with GST 4-4. The catalytic activity of pi GST 7-7 was intermediate to mμ and alpha GSTs. It is suggested that GST 3-3 may play an important role in inactivation of AFB 1-epoxide generated in vivo in the rat.

Purified glutathione s-transferases from parasites as candidate protective antigens

Sharp P. J., Smith D. R. J., Bach W., Wagland B. M. and Cobon G. S. 1991. Purified glutathione S-transferases from parasites as candidate protective antigens. International Journal for Parasitology 21: 839–846. Glutathione S-transferase (GST) activity was detected in larvae of the Australian sheep blowfly Lucilia cuprina, and in the nematode Haemonchus contortus. A specific inhibitor of the enzyme was shown to affect survival of both species of parasite in vitro. GST from both parasites has been purified and partially characterized. Antisera raised to the purified enzymes were shown to inhibit the enzyme activity in vitro. However, the antisera had no effect on the survival of either parasite.

Purification and characterization of glutathione S-transferases from rat pancreas

Glutathione S-transferases (GSTs) of rat pancreas have been characterized and their interrelationship with fatty acid ethyl ester synthase (FAEES) has been studied. Seven GST isozymes with p I values of 9.2, 8.15, 7.8, 7.0, 6.3, 5.9 and 5.4 have been isolated and designated as rat pancreas GST suffixed by their p I values. Structural, immunological and kinetic properties of these isozymes indicated that GST 9.2 belonged to the α class, GST 7.8, 7.0, 6.3 and 5.9 belonged to the μ class, whereas GST 8.15 and 5.4 belong to π class. The N-terminal sequences and p I values of the μ class isozymes suggested that rat GST subunits 3, 4 and 6 may be expressed in pancreas. N-Terminal sequences of both the π class isozymes, GST 8.15 and 5.4, were similar to that of GST-P, but there were significant differences in the substrate specificities of these two enzymes. Results of peptide finger print studies also indicated minor structural differences between these two isozymes. None of the GST isozymes of rat pancreas expressed FAEES activity. Rat pancreas had a significant amount of FAEES activity, but it segregated independently during the purification of GST indicating that these two activities are expressed by different proteins and are not related as suggested previously.

Schistosoma mansoni: Glutathione S-transferase-catalyzed detoxication of dichlorvos

Dialyzed cytosol of adult Schistosoma mansoni worm pairs catalyzed the glutathione-dependent O-demethylation of dichlorvos (2,2-dichlorovinyl dimethylphosphate), the active form of the antischistosomal drug metrifonate, to form a thioether conjugate, S-methylglutathione, and desmethyl dichlorvos. The reaction rate was dependent on both time and protein concentration, and no product was formed when either dichlorvos or glutathione was omitted from the reaction mixture. Female worm cytosols were about 2.5-fold more active per milligram of protein that those of males. Partial purification of glutathione S-transferases from male worms by affinity chromatography on glutathione-agarose showed that the reaction could be catalyzed by a preparation containing the three major isoenzymes, but that the unbound fraction, which contains at least one additional form of the enzyme that is particularly active with epoxide substrates, was 16-fold more active toward dichlorvos than the bound fraction. S-Methylglutathione also was formed by S. mansoni worm pairs incubated in the presence but not in the absence of dichlorvos. Because GSH S-transferase-catalyzed metabolism of dichlorvos results in the formation of desmethyldichlorvos, which unlike the parent compound is not an effective acetylcholinesterase inhibitor, the reaction represents a pathway of detoxication in schistosomes. It is the first example of a clinically used schistosomicide shown to be detoxicated by a conjugation pathway. These results raise the possibility that dichlorvos detoxication by S. mansoni may help explain why this species is normally refractory to metrifonate.

Independent segregation of glutathione S-transferase and fatty acid ethyl ester synthase from pancreas and other human tissues

Glutathione S-transferase (GST) isoenzymes of human pancreas were purified, characterized and evaluated for their possible role in the metabolism of ethanol. Human pancreas has at least two GST isoenzymes belonging to the Alpha class (pI 8.8 and 8.1), one belonging to the Mu class (pI 6.4) and one belonging to the Pi class (pI 4.9). During the purification of GSTs from pancreas as well as from heart, liver, lung, brain and muscle, the fatty acid ethyl ester synthase (FAEES) activity was monitored in order to evaluate the role of GSTs in metabolism of ethanol, as suggested in earlier studies. Both t.l.c. and h.p.l.c. were used to identify ethyl oleate in reaction mixtures to monitor FAEES activity. During the purification of GSTs with the use of affinity chromatography on GSH linked to epoxy-activated Sepharose 6B, FAEES and GST activities from each of these tissues segregated independently. Purified GST isoenzymes from these tissues did not exhibit any FAEES activity. Antibodies raised against Pi-class GST, as expected, immunoprecipitated most of the GST activity of brain and heart without precipitating FAEES activity. These results suggest that human GST isoenzymes belonging to the Alpha, Mu and Pi classes do not express FAEES activity. The independent segregation of GST and FAEES activities was further demonstrated by monitoring GST activity during the purification of FAEES from pancreas. It was found that purified FAEES had no GST activity towards 1-chloro-2,4-dinitrobenzene and a number of other electrophilic substrates. Results of these studies demonstrate that FAEES and GSTs are distinct proteins.

Stereoselective Glutathione Conjugation by Subcellular Fractions and Purified Glutathione S-Transferases

Stereoselective Glutathione Conjugation by Subcellular Fractions and Purified Glutathione S-Transferases

Synthesis and use of an isoform-specific affinity matrix in the purification of glutathione S-transferases from the housefly, Musca domestica (L.)

Glutathione may be linked to an agarose matrix which has been activated by treatment with epichlorhydrin. The resulting resin displayed group selectivity for the glutathione S-transferases of the housefly Musca domestics (L). The isoenzymes of low isoelectric point, which have little activity with substrates other than 1-chloro-2,4-dinitrobenzene, bound strongly to this matrix and were eluted with 10 mm glutathione at pH 7.4. On the other hand, the group of isoenzymes of higher isoelectric point, showing activity with other substrates such as 3,4-dichloronitrobenzene, did not bind. These isoenzymes did bind to a sulfobromophthalein-glutathione conjugate immobilized on agarose and could be eluted with 5 mm sulfobromophthalein at pH 7.4. The immobilized glutathione resin bound rat liver glutathione S-transferase subunits from all three molecular weight classes.

Glutathione S-transferase isoenzymes in human bronchoal veolar lavage: a possible early marker for the detection of lung cancer

The levels of glutathione S-transferase (GST) isoenzymes, GST pi, B1, B2 and mu were measured, by radioimmunoassay, in human bronchoalveolar lavage fluid from a series of patients presenting with neoplastic (n = 12) and non-neoplastic lung diseases (n = 10). Lavage fluid was obtained from the suspected abnormal area of lung and a presumed normal area of lung at the time of bronchoscopy. Concentrations of GST B1 and GST B2 were found to be significantly raised (P less than 0.02) in the lavage fluid obtained from the suspected abnormal areas of lung compared with the presumed normal area of lung, in patients later diagnosed as having cancer of the bronchus. The findings of the radioimmunoassay, of greater levels of GST B1 and B2 than GST pi in lavage fluid, were confirmed by a one-step purification of GST from lung lavage, using affinity chromatography, followed by their identification using SDS-polyacrylamide gel. We conclude that measurement of GST B1 or GST B2 in lung lavage fluid could be a useful aid in the diagnosis of lung malignancy.

Glutathione S-transferase in human bile

Glutathione S-transferase (GST) isoenzymes have been measured by specific radioimmunoassay in human bile samples. GST Mu was found in 50% of samples while GST Pi, GST B, and GST B 2 were present in all samples; GST Pi constituted the major isoenzyme identified. The findings of the radioimmunoassay were confirmed by a one-step purification of GST from bile, using affinity chromatography, followed by their identification using sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE). Inhibition studies showed that, at the concentrations of bile salts found in bile, GST Pi would have little or no enzymic activity. It is proposed that GST Pi acts as a carrier protein of toxic, non-substrate, ligands to remove as yet unidentified substances from biliary epithelial cells and prevent their reabsorption.

Purification and properties of hepato-pancreas and green gland cytosolic glutathione S-transferases of the red swamp crayfish, Procambarus clarkii

Cytosolic fractions from hepato-pancreas and green gland of the red swamp crayfish, Procambarus clarkii contained high glutathione S-transferase (GST) activity, 5·4±0·6 and 4·2±0·2 μmol/min/mg protein, respectively as measured with 1-chloro-2,4-dinitrobenzene (CDNB). The GSTs have been purified to electrophoretic homogeneity from 105 000 x g supernatant of hepato-pancreas and green gland homogenates by employing Sephadex G-200, DEAE-Sephacel and chromatofocusing. Two isoenzymes were purified from hepato-pancreas; one which eluted at pH 5·2 with a specific activity of 86·8 μmol/min/mg, and one at pH 5·1 with a specific activity of 58·3 μmol/min/mg. Purification of GST from green gland resulted in two isoenzymes which eluted at pH 5·1 and 4·6 and had specific activities of 38·0 and 164 μmol/min/mg, respectively. Preliminary data suggest that all of the isoenzymes are dimers with subunit molecular weights of 22 000–23 000 Da. The purified GST were most active in the conjugation of CDNB and showed little or no activity towards other common substrates. Cytosolic activities with p-nitrophenyl acetate and p-nitrophenyl trimethylacetate were higher than could be accounted for by the activity of the purified isoenzymes. These data suggest the presence of isoenzymes in cytosol that might have gone undetected using a purification scheme which employs CDNB as the electrophilic substrate. Kinetic parameters and inhibition characteristics further distinguished between the isoenzymes.

Marine invertebrate glutathione- S-transferases: purification, characterization and induction

High glutathione- S-transferase (GST) activity was found in hepatopancreas and gill cytosol of the blue crab ( Callinectes sapidus) and the digestive gland cytosol of two marine gastropods ( Nassarius obsoletus and Cerithium floridanum). Purification of GST from crab hepatopancreas by Sephadex G-200, DEAE-Sephacel and chromofocusing resulted in the isolation of two isoenzymes with isoelectric points of 5·9 and 5·7 (GST 5·9 and GST 5·7). Antibodies were prepared to these two isoenzymes and the two forms cross-reacted immunologically. The two transferases had similar molecular weights, amino acid compositions, substrate specifities and kinetic parameters. Crab gill cytosol showed one isoenzyme which reacted with antibodies to GST 5·9 and GST 5·7. The major isoenzyme of N. obsoletus was a basic form while C. floridanum showed a homodimer acidic form. The gastropod GST forms did not react with antibodies to crab GST. The presence of the phenolic antioxidant, butylated hydroxytoluene, in the diet of blue crab or shrimp ( Penaeus aztecus) resulted in high hepatic GST activity.

Studies on the subunits of rabbit hepatic glutathione S-transferases.

We previously reported that the purified glutathione S-transferases from rabbit liver are homodimers or heterodimers of four different subunits, having molecular weight of 24500 (Y1), 25000 (Y2), 26500 (Y3) and 28000 (Y4).Their subunit compositions were also reported to be Y1Y1 (R1a), Y1Y3 (R3a), Y3Y3 (R3b), Y2Y2 (R2) and Y2Y4 (Rib, Ric, Rid).In the present study, two additional isozymes, R3o and R3c, were obtained.They showed the same subunit compositions as R la and R3b, respectively.Each subunit with the same molecular weight among different isozymes was demonstrated to have the same pI value by isoelectric focusing on polyacrylamide-gel plates containing 7.5m urea (Y1, pI 8.49;Y2, pI 7.86;Y3, pI 8.41; Y4, pI 7.15).R3a (Y1Y3) in a reconstitution experiment using 4 M guanidine hydrochloride gave Y1Y1 homodimer (R3o), Y1Y3 heterodimer (R3a) and Y3Y3 homodimer (R3b).Similarly, the reconstituted Ric (Y2Y4) gave Y2Y2 homodimer (R2), Y2Y4 heterodimer (Ric) and an isozyme which probably has the subunit composition of Y4Y4.The failure of the in vitro formation of heterodimers such as Y1Y2, Y1Y4, Y2Y3 and Y3Y4 suggests that the possible combinations of subunits are limited.

The purification and characterization of glutathione S-transferase from the hepatopancreas of the blue crab, Callinectes sapidus

High glutathione S-transferase activity was found in the cytosol of F-cells from the hepatopancreas of the blue crab ( Callinectes sapidus). Purification of glutathione S-transferase from hepatopancreas extracts by Sephadex G-200, DEAE-Sephacel, and chromatofocusing resulted in the isolation of two isozymes with isoelectric points of 5.9 and 5.7, as determined by analytical isoelectric focusing. Using 1-chloro-2,4-dinitrobenzene as the substrate the specific activities of the two purified isozymes were 222 and 182 μmol/min/mg, respectively. There was no evidence for basic transferase isozymes. In addition to 1-chloro-2,4-dinitrobenzene the purified glutathione S-transferase isozymes showed activity with p-nitrophenyl acetate, p-nitrobenzyl chloride, bromosulfophthalein, and benzopyrene oxide. Thus, both substitution and addition reactions associated with vertebrate glutathione S-transferase were found in the crab transferases. There was no activity when ethacrynic acid, methyl iodide, trans-4-phenyl-3-buten-2-one, 1,2-epoxy-( p-nitrophenoxy)propane, cumene hydroperoxide, and t-butyl hydroperoxide were used as substrates. The lack of peroxidase activity is of interest since this activity is commonly found in vertebrate transferase isozymes. The two transferases had a dimeric M r of 40,800 with similar amino acid compositions and similar kinetic parameters ( V max, K m, and pH maxima) with 1-chloro-2,4-dinitrobenzene as substrate. The two transferases could be distinguished by their isoelectric points, molecular mass of the monomers (22,300 for GST 1 and 22,300 and 22,400 for GST 2), and different inhibitor mechanisms with hematin and bromosulfophthalein.

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

1. 1. Extracts prepared from liver, kidney, lung and brain of camel contain glutathione, glutathione S-transferase and glutathione reductase. 2. 2. Liver had the highest level of glutathione (218.7 μmol/g wet weight) whereas brain had the lowest level (66.4 μmol/g wet weight). 3. 3. The highest activity for glutathione reductase was found in the kidney (2.6 μmol/min/mg protein) while the lowest activity was found in the lung (0.9 μmol/min/mg protein). 4. 4. Glutathione S-transferase activity was the highest in liver (4.2 μmol/min/mg protein) and the lowest in brain (1 μmol/min/mg protein). 5. 5. 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.

New assay procedure for glutathione S-transferase using analytical capillary isotachophoresis: A procedure for the simultaneous analysis of reduced and oxidized glutathione together with glutathione conjugates of electrophilic compounds

A procedure for the simultaneous analysis of reduced and oxidized glutathione together with glutathione conjugates of electrophilic organic compounds by capillary isotachophoresis is described. This method can be applied to quality control of such synthetic conjugates, which are commonly employed as affinity ligands in the chromatographic purification of glutathione S-transferases (EC 2.5.1.18). Using this new assay procedure, it has been found that the glutathione conjugate of 1-chloro-2,4-dinitrobenzene is unstable in neutral to alkaline solution, degrading to glutathione and 2,4-dinitrophenol. Anomalies in the electrophoretic mobilities of a homologous series of alkylglutathione derivatives were observed. A possible explanation is provided on the basis of the formation of micelle-like aggregates, this tendency increasing with increasing length of the hyrocarbon chain. A useful and widely applicable aspect of this analytical procedure is as a method of monitoring glutathione S-transferase. The major advantages of this new assay technique can be summarized as follows: (1) the assay does not depend on optical properties of the acceptor, (2) the reaction can be monitored in the presence of unrefined biological material;aliquots can be injected without pretreatment and (3) the spontaneous oxidation of glutathione in the assay mixture can be monitored simultaneously.