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JOURNAL OF BACTERIOLOGY, Nov. 1989, P. 6039-60420021-9193/89/116039-04$02.00/0Copyright C 1989, American Society for MicrobiologyVol. 171, No. 11Purification and Some Properties of Glutathione S-Transferase fromEscherichia coli BMASAKI IIZUKA, YOSHIHARU INOUE,* KOUSAKU MURATA, AND AKIRA KIMURAResearch Institute for Food Science, Kyoto University, Uji, Kyoto 611, JapanReceived 31 January 1989/Accepted 9 August 1989Glutathione S-transferase was purified approximately 2,300-fold from cell extracts of Escherichia coli B witha 7.5% activity yield. The molecular weight of the enzyme was 45,000, and the enzyme appeared to consist oftwo homogeneous subunits. The enzyme was almost specific to 1-chloro-2,4-dinitrobenzene (K,. 1.43 mM) andglutathione (K,,, 0.33 mM). The optimal pH and optimal temperature for activity were 7.0 and 50°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 ,umol/min per mg of protein) was significantly lower than those of the enzymes frommammals, plants, and fungi.Glutathione S-transferases (GSTs) constitute an importantclass of detoxifying enzymes. The enzyme catalyzes theconjugation of glutathione (GSH) with various compoundshaving electrophilic and/or hydrophobic sites (6). The enzyme is thought to protect the cells against foreign compounds such as pesticides, drugs, and carcinogens (7). Theenzymes have been extensively purified from mammals suchas human (8), mouse (12), cattle (3), and rat (21), and theirproperties have been characterized in detail. However, thedata on microbial GSTs are largely lacking, and the enzymehas been purified only from Fusarium oxysporum f. sp.melonis (5), Mucorjavanicus (1), and Tetrahymena thermophila (18). Although attempts to detect GST activity in cellextracts of bacteria (2, 11, 21), yeasts (4, 9), mold (1, 5), andprotozoa (18) have been made, these results are fragmentaryand not enough to compare the properties of microbialenzymes with those of enzymes from mammals. Here wereport the purification and partial characterization of GSTfrom Escherichia coli B.MATERIALS AND METHODSChemicals. GSH was purchased from Kohjin Co., Ltd.,Tokyo, Japan. 1-Chloro-2,4-dinitrobenzene was from TokyoKasei Kogyo Co., Ltd., Tokyo, Japan. Glutathione agarosewas prepared from AH-Sepharose CL-4B according to thevendor’s specifications (Pharmacia).Assays. The activity of GST was assayed essentially by themethod of Habig et al. (6). The reaction mixture (1.0 ml)consisted of 0.1 M potassium phosphate buffer (KPB) (pH7.0), 1.0 mM EDTA, 1.0 mM GSH, 1.0 mM 1-chloro-2,4-dinitrobenzene (CDNB), and enzyme. The enzyme activity was calculated by using a molar extinction coefficientof S-(2,4-dinitrophenyl)glutathione as 9.6 M-1 cm-‘ at 340nm and 25°C. One unit of enzyme activity was defined as theamount producing 1 nmol of conjugate of GSH with CDNBper min. Protein was determined by the method of Lowry etal. (13). GSH in cells was determined by the method ofMurata et al. (17).Growth experiments. In order to investigate the effect ofelectrophiles on the formation of GST activity, cells of E.coli B were grown in a test tube containing 5 ml of nutrientmedium (1.0% glucose, 0.1% yeast extract, 1.0% peptone,0.5% meat extract, 0.1% MgSO4- 7H20, and 0.5% K2HPO4* Corresponding author.[pH 7.0]) with reciprocal shaking at 37°C for 20 h. The cellswere transferred into a 2-liter Sakaguchi flask containing 500ml of the same fresh medium, and the initial turbidity(OD610) was adjusted to 0.03. Electrophilic compounds suchas CDNB and o-dinitrobenzene (o-DNB), which were dissolved in a minimal amount of acetone, were then added tothe culture medium described above. E. coli B cells werecultivated at 37°C with reciprocal shaking, and the growth ofcells was monitored by measuring the turbidity at 610 nm.An appropriate amount of cells was withdrawn, washed oncewith 0.85% NaCl solution, and then suspended in a smallamount of 10 mM KPB (pH 7.0) containing 1.0 mM EDTA,0.2 mM GSH, and 20% (wt/vol) glycerol (buffer A). The cellswere disrupted for 5 min at 90 kHz and 0°C on a KubotaInsonator (model 200M). The homogenates were centrifugedat 25,000 x g for 20 min at 4°C, and the supernatants wereused as the source of GST.Purification of GST. (i) Cell extracts. Cells (187 g, wetweight) from a 30-liter nutrient culture were washed oncewith 0.85% saline solution and suspended in 600 ml of bufferA. The cells were disrupted as shown before and thencentrifuged at 25,000 x g for 30 min at 4°C. Unless otherwisestated, all purification procedures were done at 0 to 4°C.(ii) Ammonium sulfate fractionation. Solid ammonium sulfate (146 g, 45% saturation) was added to the crude extracts(24,144 mg, 600 ml), and the mixture was stirred for 2 h. Theprecipitates were removed by centrifugation (15,000 rpm for30 min). Another 80 g of ammonium sulfate was added to theresultant supernatants, and the precipitates formed werecollected by centrifugation (15,000 rpm for 20 min). Thepellets were resolved in 250 ml of buffer A.(iii) Butyl Toyopearl 650M column chromatography. Theenzyme solution (3,278 mg of protein, 260 ml) was brought to30% saturation by adding ammonium sulfate, and the mixture was applied to a Butyl Toyopearl 650 M column (4.5 by60 cm) equilibrated with buffer A containing 30% ammoniumsulfate. The enzyme was eluted with a linear gradient ofammonium sulfate (30 to 0%; total volume, 1,600 ml).Fractions were collected as 16-ml portions every 14 min. Theactive fractions (conductivity, 8.4 to 7.9 mS) were pooledand dialyzed against buffer A overnight.(iv) Hydroxylapatite column chromatography. The dialysate (108.7 mg of protein, 205 ml) was loaded on a hydroxylapatite column (2.8 by 9 cm) equilibrated with buffer A.The enzyme was eluted with a linear gradient of KPB (pH6039Downloaded from on February 24, 2021 by guest6040 IIZUKA ET AL.7.0) (10 to 300 mM; total volume, 400 ml) containing 1.0 mMEDTA, 0.2 mM GSH, and 20% glycerol. Fractions werecollected at a flow rate of 5 ml/5 min. The active fractions(conductivity, 0.24 to 0.54 mS) were combined and concentrated to approximately 9 ml with an Amicon PM10 membrane.(v) Sephadex G-150 column chromatography. The concentrate (67.3 mg of protein, 9 ml) was applied to a SephadexG-150 column (1.4 by 90 cm) equilibrated with buffer A.Elution of the proteins was performed with buffer A at a flowrate of 3 ml/15 min. The active fractions (nos. 35 to 41) werepooled and concentrated as before.(vi) DEAE-Sepharose CL-6B column chromatography. Theconcentrate (9.02 mg of protein, 6 ml) was applied to aDEAE-Sepharose CL-6B column (1.4 by 47 cm) equilibratedwith buffer A. The enzyme was eluted with a linear gradientof KPB (50 to 300 mM; total volume, 400 ml) containing 1.0mM EDTA, 0.2 mM GSH, and 20% glycerol at a flow rate of4 ml/10 min. The active fractions (conductivity, 0.9 to 1.0mS) were pooled and dialyzed against 10 mM KPB (pH 7.0)overnight.(vii) GSH agarose affinity column chromatography. Thedialysate (4.46 mg of protein, 12 ml) was applied to a GSHagarose affinity column (1.4 by 7.8 cm) equilibrated with 10mM KPB (pH 7.0). The column was washed first with 200 mlof the same buffer containing 0.1 M KCl and then with 200 mlof 10 mM KPB (pH 7.0). The adsorbed enzyme was elutedwith 10 mM KPB (pH 7.0) containing 10 mM GSH. Theactive fractions were pooled and dialyzed against 10 mMKPB (pH 7.0) overnight. The dialysate was stored at -20°Cuntil use.Molecular weight determination. The molecular weight ofthe enzyme was estimated by gel filtration of a SephadexG-150 column (1.4 by 90 cm) by the method of Andrews (2).The enzyme was eluted with 10 mM KPB (pH 7.0), and theeluates were collected at a flow rate of 3 ml/15 min.Polyacrylamide gel electrophoresis. Polyacrylamide gelelectrophoresis in the presence of sodium dodecyl sulfate(SDS) was conducted by the method of Laemmli (10). Thegel was stained for proteins with Coomassie brilliant blue.RESULTSEffect of electrophilic compounds on the formation of GST.The effect of CDNB or o-DNB on the formation of GSTactivity was examined. Without the electrophilic compounds, GST activity was formed in association with growth(Fig. 1A). When CDNB (0.2 mM; Fig. 1B) or o-DNB (0.2mM; Fig. 1C) was added to the culture, the growth of cellswas initially repressed. However, no increase in GST activity over that of control cells grown in the absence of thesechemicals (Fig. 1A) was observed. Cellular GSH contentwas not affected in the presence or absence of electrophiliccompounds. The content increased in association with cellgrowth, reached a maximum at stationary phase, and thendeclined.Purification of GST. The overall purification proceduresfor GST are summarized in Table 1. The enzyme waspurified approximately 2,300-fold, with 7.5% activity yield.The purified enzyme migrated as a single protein band witha molecular weight of 25,000 on SDS-polyacrylamide gelelectrophoresis (Fig. 2B). On the other hand, the molecularweight of the purified enzyme was estimated to be 45,000 bya calibrated column of Sephadex G-150 (Fig. 2A).Substrate specificity. The purified enzyme was most activeon CDNB as an electrophilic substrate, and the Km value.-I)10c0 050co0OFa .f r 0-, 10 Jo10 20 10 20 IO 20Time (h)FIG. 1. Effect of electrophiles on GST activity and GSH content.The cells were cultivated at 37°C with reciprocal shaking in theabsence of electrophiles (A) or in the presence of 200 ,uM CDNB (B)or 200 FM o-DNB (C). Growth (OD610) (0), GST activity (units permilligram of protein) (@), and GSH content (micrograms per gram ofcells) (A) were measured as described in Materials and Methods.was calculated to be 1.43 mM. The relative activities for1-nitro-3,4-dichlorobenzene and p-nitrobenzylchloride wereonly 4 and 2%, respectively, when the activity towardCDNB was taken as 100% (Table 2). Other electrophiliccompounds such as 4-nitropyridine-N-oxide, 1,2-epoxy-3-(p-nitrophenoxy)propane, and p-nitrophenylbromide wereinert as substrates. The enzyme utilized GSH most efficiently as a nucleophilic substrate (relative activity, 100%).Other thiol compounds, such as L-cysteine (6%), -y-L-glutamyl-L-cysteine (35%), dithiothreitol (35%), and 2-mercaptoethanol (22%), were also found to be utilized as substrates,but with lower efficiencies (Table 2). The apparent Km valuefor GSH was calculated to be 0.33 mM.Effect of pH and temperature. The enzyme was most activeat pH 7.0 (Fig. 3A) and was stable between pH 5 and 11 (Fig.3B). The optimal temperature for activity was ca. 50°C. Theactivity of the enzyme was stable up to 50°C, and the activitywas rapidly lost above 50°C.Effect of various chemicals on GST activity. The effects ofmetal ions and sulfhydryl-blocking agents on the enzymeactivity were investigated (Table 3). The enzyme activitywas inhibited by Fe2″ and Zn2+ at 1.0 mM, the inhibitionbeing 47 and 61%, respectively. Other bivalent metal ionssuch as Ca2″, Mg2+, Co2+, Mn2+, and Ni2+ (1.0 mM)showed no appreciable inhibitory effects on the enzymeactivity. On the other hand, the enzyme activity was reducedabout 30% by iodoacetamide at 5.0 mM. N-Ethylmaleimide(5.0 mM) and p-chloromercuribenzoate (1.0 mM) showed noeffects on activity of the enzyme.DISCUSSIONKumagai et al. showed that the GST in the yeast Issatchenkia orientalis is induced in the presence of o-DNB orTABLE 1. Purification of GST from E. coli B ProteinTotalSp act YedPurifiStepStep(mg) (gactivity (U) (U/mg protein) of Yilcation (fold)Cell extract(NH4)2SO4 (45-65%)Butyl-Toyopearl 650MHydroxylapatiteSephadex G-150DEAE-SepharoseCL-6BGlutathione agarose24,1443,278108.767.333,60012,2001.39 1003.72,40577.325.020.56,890 102.49.02 4,608 510.64.46 3,072 668.913.7 367.39.1 495.60.78 2,520 3,2147.5 2,314 J. BACTERIOL.Downloaded from on February 24, 2021 by guestGLUTATHIONE S-TRANSFERASE FROM E. COLI B 6041(A)7 – a5iwb TX037eC_iCd1 2 3(B)– 97K-68K-43K* -29K-18KVe/Voa bFIG. 2. Molecular weight determination. (A) The molecularweight (M.W.) of the enzyme was determined on a calibratedcolumn of Sephadex G-150 as described in Materials and Methods.Standard proteins used: a, bovine serum albumin (M, 68,000); b,ovalbumin (Mr 43,000); c, a-chymotrypsinogen (M, 25,700); and d,lysozyme (M, 14,300). The position of GST is indicated by a solidcircle. VO and Ve are void volume of column and elution volume ofproteins, respectively. Void volume of the column was determinedby using blue dextran. (B) The purified enzyme (10 ,g) was treatedwith SDS in the presence of dithiothreitol and electrophoresed on a0.1% SDS-12.5% polyacrylamide gel. Lane a, GST; lane b, molecular weight markers (from the top: phosphorylase b [M, 97,400];bovine serum albumin [Mr 68,000]; ovalbumin [Mr 43,000]; carbonicanhydrase [Mr 29,000]; and ,-lactoglobulin [Mr 18,400]).L-cysteine and glycine in the culture (9). Cohen et al.reported that GSH content in the mycelia of Fusariumoxysporum and Rhizoctonia solani was initialy reduced inthe presence of electrophilic compounds such as pentachloronitrobenzene and CDNB and then increased gradually tothe original level (5). GST activity of the electrophile-treatedmycelia of F. oxysporum and R. solani also decreases almostin parallel the intracellular GSH content. However, suchelectrophilic compounds showed little effects on both GSTformation and GSH content in E. coli B. This was presumably due to the impermeability of the electrophiic compounds across the cell membrane. The addition of L-cysteineor glycine also showed no effect on GST formation (data notshown).TABLE 2. Substrate specificity of GST from E. coli BActivitya SubstrateConcn(mM)(iLmol/minper mg ofprotein) ElectrophilesCDNB 1.0 3.2 (100)1-Nitro-3,4-dichlorobenzene 1.0 0.13 (4)p-Nitrobenzylchloride 1.0 0.06 (2)4-Nitropyridine-N-oxide 0.5 NDb1,2-Epoxy-3-(p-nitrophenoxy)propane 5.0 NDp-Nitrophenylbromide 1.0 NDThiolsGSH 1.0 3.2 (100)-y-L-Glutamyl-L-cysteine 1.0 1.1 (35)L-Cysteine 1.0 0.2 (6)Dithiothreitol 1.0 1.1 (35)2-Mercaptoethanol 1.0 0.7 (22)a Numbers in parentheses indicate relative activity compared with CDNB(electrophiles) or GSH (thiols).b ND, Not detected.a 50 50 04~~~~~~~~~~~~~~~~~~~~~~~~~~c.3 5 7 9 11 3 5 7 9pHFIG. 3. Effect of pH on activity and stability of GST. (A) Theactivity of the enzyme was determined in the standard assayconditions described in Materials and Methods, except for thebuffer. The buffers used were (A) sodium acetate buffer (pH 3 to 6),(0) potassium phosphate buffer (pH 6 to 8), and (0) Tris hydrochloride buffer (pH 6 to 9), and the activity at pH 7.0 in potassiumphosphate buffer was taken as 100%. (B) Purified enzyme (1 ,ug) wasincubated in various buffers (100 mM; total volume, 100 ,u) at 40Cfor 12 h, and then 20 ,ul of the mixture was transferred into thestandard assay mixture to determine residual activity. Buffers usedwere (A) sodium acetate buffer (pH 3 to 6), (0) potassium phosphatebuffer (pH 6 to 8), (0) Tris hydrochloride buffer (pH 6 to 9), and (O)glycine-KOH buffer (pH 8 to 11), and the residual activity at pH 7.0in potassium phosphate buffer was taken as 100%.Based on the above results, electrophiles were not addedto the nutrient medium when enzyme purification was carried out. The GST of E. coli B was purified to the homogeneous state by SDS-polyacrylamide gel electrophoresis(PAGE). The molecular weight of the purified GST from E.coli B was calculated to be 45,000 on gel filtration ofSephadex G-150 column chromatography. PAGE in thepresence of SDS gave a subunit molecular weight of 25,000.The E. coli B enzyme seemed to be similar to those ofmammalian and plant and mold (Mucor javanicus [1]) enzymes, all of which are dimers with a molecular weight of40,000 to 60,000, but was different from the enzyme ofTetrahymena thermophila (18). The GST of the organism isa monomer with a molecular weight of 33,000 to 35,000. Themolecular weight of the native GST of F. oxysporum isTABLE 3. Effect of various chemicals on GST activityActivitya AdditionConcn (mM)(,umol/min per mg ofprotein)MetalsNone3.2 (100)FeCl21.01.5 (47)ZnCl21.02.0 (61)MgCl21.02.8 (87)CaCl21.03.2 (100)CoCl21.02.7 (84)MnCl21.03.3 (104)NiC121.03.6 (114)Sulfhydryl-blocking agentsNone3.2 (100)3.1 (97)2.2 (69)3.1 (97) benzoate a Numbers in parentheses indicate relative activity compared to controls(no addition).VOL. 171, 1989Downloaded from on February 24, 2021 by guest6042 IIZUKA ET AL.obscure, although the molecular weight of 25,000 has beenobtained by SDS-PAGE (5). The specific activity for CDNBof the purified E. coli enzyme was 3.24 ,umol/min per mg ofprotein, whereas the GSTs purified from other sources, suchas mammals (mouse [104 to 281 p.mol/min per mg of protein),cattle [13 to 15 ,umol], human [16 to 37 pLmol], and rat [8.3 to31.4 ,umol] [8, 12, 3, 19]), plants (corn [21 to 66 ,umol] [16],fungi (F. oxysporum [35.4 ,umol], and M. javanicus [13.0,umol] [1, 5]), and protozoa (T. thermophila [35.0 ,umol] [18])had higher enzyme activities than E. coli. For mammalianand plant enzymes the enzyme activity values vary depending on the number of isozymes. In E. coli B cell, the GSTwas not separated into more than one active peak duringpurification, suggesting that E. coli B did not contain isozymes (5). The isozyme has not been reported for molds (5),although Casalone et al. have suggested that the differencesin GST activity observed in the various yeast strains aroselargely from the difference in the isozyme composition (4).Morgenstern et al. have shown that the enzyme purifiedfrom rat liver microsome was stimulated eightfold by thetreatment with N-ethylmaleimide and fourfold with iodoacetamide. On the other hand, the soluble enzyme of ratliver cytoplasm was not affected by treatment with suchsulfhydryl-blocking agants (15). In our study, the activity ofthe E. coli B enzyme was lost approximately 30% when itwas treated with iodoacetamide, while N-ethylmaleimideand p-chloromercuribenzoate showed no appreciable effects. The results suggested that the E. coli enzyme containsno active-site thiol.The purified GST from E. coli shows neither seleniumdependent nor independent glutathione peroxidase activitywhen H202, cumenehydroperoxide, or tert-butylhydroperoxide was used as a substrate (data not shown). On the otherhand, GSTs purified from rat and mouse livers exhibitedselenium-independent glutathione peroxidase activity (19,22), indicating that the properties of catalytic sites betweenmammalian and microbial enzymes may be different. ThecDNAs responsible for GSTs of corn (GST III [14]) and rat(Ya and Yc [21]) have been cloned, sequenced, and expressed in E. coli cells. The molecular cloning of the GSTgene of E. coli is now in progress in order to elucidate thedifference in the molecular structure between the mammalian and E. coli enzymes.LITERATURE CITED1. Ando, K., M. Honma, S. Chiba, S. Tahara, and J. Mizutani.1988. Glutathione transferase from Mucor javanicus Agric.Biol. Chem. 52:135-139.2. Andrews, P. 1965. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J.%:595-606.3. Asaoka, K. 1984. Affinity purification and characterization ofglutathione S-transferase from bovine liver. J. Biochem. 95:685-696.4. Casalone, E., C. D. Llio, G. Federici, and M. Polsinelli. 1988.Glutathione and glutathone metabolizing enzymes in yeast. J.Microbiol. 54:367-375.5. Cohen, E., A. Gamliel, and J. Katan. 1986. Glutathione andglutathone S-transferase in fungi: effect of pentachloronitrobenzene and 1-chloro-2,4-dinitrobenzene: purification andcharacterization of the transferase from Fusarium. Pestic. Biochem. Physiol. 26:1-9.6. Habig, W. H., M. J. Pabst, and W. B. Jakoby. 1974. GlutathioneS-transferase: the first enzymatic step in mercaptric acid formation. J. Biol. Chem. 249:7130-7139.7. Huston, D. H. 1976. In D. D. Kaufman, G. G. Still, G. D.Paulson, and S. K. Bandal (ed.), Bound and conjugated pesticide residues, p. 103-131. American Chemical Society, Washington, D.C.8. Kamisaka, K., W. H. Habig, J. N. Ketley, I. M. Arias, and W. B.Jakoby. 1975. Multiple forms of human glutathione S-transferase and their affinity for bilirubin. Eur. J. Biochem. 60:153-161.9. Kumagai, H., H. Tamaki, Y. Koshino, H. Suzuki, and T.Tochikura. 1988. Distribution, formation and stabilization ofyeast glutathione S-transferase. Agric. Biol. Chem. 52:1377-1382.10. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.11. Lau, E. P., L. Niswander, D. Watson, and R. Ray Fail. 1980.Glutathione S-transferase is present in a variety of microorganisms. Chemosphere 9:565-569.12. Lee, C.-Y., L. Johnson, R. H. Cox, J. D. McKinney, and S.-M.Lee. 1981. Mouse liver glutathione S-transferase: biochemicaland immunological characterization. J. Biol. Chem. 256:8110-8116.13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.14. Moore, R. E., M. S. Davies, K. M. O’Conell, E. I. Harding, R. C.Wiegand, and D. C. Tiemeier. 1986. Cloning and expression of acDNA encoding a maize glutathione-S-transferase in E. coli.Nucleic Acids Res. 14:7227-7235.15. Morgenstern, R., J. W. Depierre, and L. Ernster. 1979. Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents. Biochem. Biophys. Res. Commun. 87:657-663.16. Mozer, T. J., D. C. Tiemeier, and E. G. Jaworski. 1983.Purification and characterization of corn glutathione S-transferase. Biochemistry 22:1068-1072.17. Murata, K., K. Tani, J. Kato, and I. Chibata. 1980. Excretion ofglutathione by methylglyoxal-resistant Escherichia coli. J. Gen.Microbiol. 120:545-547.18. Overbaugh, J. M., P. E. Lau, V. A. Marino, and R. Fail. 1988.Purification and preliminary characterization of amonomericglutathione S-transferase from Tetrahymena thermophila. Arch.Biochem. Biophys. 261:227-234.19. Scott, T. R., and R. E. Kirsch. 1987. The isolation of fetal ratliver glutathione S-transferase isozymes with high glutathioneperoxidase activity. Biochim. Biophys. Acta 926:264-269.20. Shishido, T. 1981. Glutathione-S-transferase from Escherichiacoli. Agric. Biol. Chem. 45:2951-2953.21. Telakowski-Hopkins, C. A., J. A. Rodkey, C. D. Bennett,A. Y. H. Lu, and C. B. Pickett. 1985. Rat liver glutathioneS-transferases: construction of a cDNA clone complementary toa Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit. J. Biol. Chem. 160:5820-5825.22. Warholm, M., H. Jensson, M. K. Tahie, and B. Mannervik.1986. Purification and characterization of three distinct glutathione transferases from mouse liver. Biochemistry 25:4119-4125.J. BACTERIOL.Downloaded from on February 24, 2021 by guest


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