Vol. 171, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1989, p. 1173-11770021-9193/89/021173-05$02.00/0Copyright © 1989, American Society for Microbiology

Purification and Properties of Glutathione Transferase fromIssatchenkia orientalis

HISANORI TAMAKI, HIDEHIKO KUMAGAI,* AND TATSUROKURO TOCHIKURA

Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

Received 24 October 1988/Accepted 17 November 1988

Glutathione transferase (GST) (EC 2.5.1.18) was purified from a cell extract of Issatchenkia orientalis, andtwo GST isoenzymes were isolated. They had molecular weights of 37,500 and 40,000 and were designated GSTY-1 and GST Y-2, respectively. GST Y-1 and GST Y-2 gave single bands with molecular weights of 22,000 and23,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GST Y-1 and GST Y-2were immunologically distinguished from each other. GST Y-1 showed specific activity 10.4-times and6.0-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 bromosul-fophthalein and trans-4-phenyl-3-buten-2-one were used. GST Y-1 and GST Y-2 hjd Km values of 0.51 and 0.75mM for glutathione, respectively, and of 0.16 and 4.01 mM for i-chloro-2,4-dinitrobenzene. GST Y-1 was

significantly inhibited by Cibacrqn blue 3G-A, and GST Y-2 was significantly inhibited by bromosulfophtha-lein.

Glutathione transferase (GST) catalyzes the conjugationof various electrophilic substrates, including xenobioticssuch as carcinogens and mutagens, with glutathione (GSH).In the mammalian liver, these GSH conjugates are metabo-lized further by y-glutamyl transpeptidase, cysteinylglyci-nase, and N-acetyl transferase through the mercapturic acidbiosynthesis pathway (7). The distribution of GST has beenreported from bacteria to mammals (14), and many GSTisoenzymes have been isolated and characterized in variousmammals (2, 3, 9, 16, 20). Some GSTs have been reported tohave alternative functions other than detoxification, such asthe reduction of organic hydroperoxides by GSH peroxidaseactivity (19) and the possible formation of leukotriene C fromleukotriene A (5).GSH was first isolated from yeasts and is abundant in

yeast cells (26), but there are few reports of yeast GST (15,18), and GST has not been purified from yeast cells to ahomogeneous state. Recently we reported the distribution ofGST in yeasts and the formation and the stabilization ofGSTin Issatchenkia orientalis, which showed the highest GSTactivity of all yeast strains investigated (16a).

In this study, GST was purified from I. orientalis andcharacterized to determine the physiological role of GSHand GST in yeast cells.

MATERIALS AND METHODSReagents. Bromosulfophthalein, 1,2-epoxy-3-(p-nitrophe-

noxy)-propane, ethacrynic acid, and hematin were pur-chased from Sigma Chemical Co. 1-Chloro-2,4-dinitroben-zene (CDNB), o-dinitrobenzene (o-DNB), p-nitrobenzoylchloride, p-nitrophenethyl bromide, and DEAE-cellulosewere from Wako Pure Chemical Co. 4-Nitropyridine-N-oxide was from Nakarai Chemical Co. 1,2-Dichloro-4-ni-trobenzene, tributyltin acetate, and triphenyltin chloridewere from Tokyo Kasei Co. trans-4-Phenyl-3-buten-2-onewas from Aldrich Chemical Co., and Cibacron blue 3G-Awas from Fluka AG. Sephadex G-100, Phenyl-SepharoseCL-4B, CM-Sepharose CL-4B, PBE 94, and Polybuffer 96were from Pharmacia. Reduced GSH was a generous gift

* Corresponding author.

from Kirin Brewery Co. Other chemicals were purchasedfrom commercial sources.

Yeast strain and culture. I. orientalis was obtained fromstock cultures of the Laboratory of Industrial Microbiology,Department of Food Science and Technology, Faculty ofAgriculture, Kyoto University, Kyoto, Japan.

Cells were picked up from a basal medium slant culturecontaining 2% glucose, 0.5% peptone, 0.05% yeast extract,0.05% KH2PO4, 0.05% K2HPO4, and 0.02% MgSO4, inocu-lated into a test tube containing 5 ml of the basal medium,and grown at 28°C overnight with reciprocal shaking. Eachtest tube culture was transferred to a 2-liter Sakaguchi flaskcontaining 500 ml of basal medium and was then grown at280C overnight with reciprocal shaking. Two such subcul-tures were inoculated into a 30-liter jar fermentor (typeMSJ-U 301, Marubishi Co.) containing 25 liters of inducingmedium consisting of 200 ,uM o-DNB, 0.1% glycine, and0.1% L-cysteine, besides the basal medium as describedbefore (16a). Cultivation was carried out .at 30°C with aera-tion (1 liter per liter of medium per min) and agitation (200rpm). The grown cells were harvested at the late-exponentialphase with a refrigerated continuous-flow centrifuge (typeGLE, Carl Padberg GmbH).Enzyme assay for GST activity. During the purification,

GST activity was assayed spectrophotometrically with thesubstrate CDNB as described previously (16a). For thesubstrate specificities, the enzyme activity was determinedby the method of Habig et al. (13). GST activity was alsodetermined by measuring nitrite released enzymatically fromo-DNB, by using the diazo-coupling method of Asaoka andTakahashi (4) with a slight modification (16a).

Protein determination. Protein concentrations were deter-mined by the method of Lowry et al. (21) with ovalbumin asa standard and by the method of Lowry modified by Bensa-doun and Weinstein (6) when the enzyme solution containedglycerol.

Electrophoresis. Disc polyacrylamide gel electrophoresis(disc PAGE) was performed by the method of Tamura andUi (25). Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) was carried out as described byLaemmli (17). Proteins on the native gel were stained with

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amido black 10J3, and those on the Laemmli gel were stainedwith Coomassie blue R-250.

Molecular weight estimation. Molecular weights were de-termined by gel filtration on a high-performance liquidchromatograph equipped with a TSK-GEL 2,000 SW column(7.5 by 30 mm) (Tosoh Co.) equilibrated with 0.05 Mpotassium phosphate (pH 7.0). Elution was performed withthe same buffer, and A280 was measured.The subunit molecular weights were estimated by SDS-

PAGE, with an LMW molecular weight calibration kitpurchased from Pharmacia.

Chromatofocusing. The enzyme solution was applied on aPBE 94 column (0.8 by 20 cm) equilibrated with 0.25 Methanolamine hydrochloride (pH 9.4) containing 20% glyc-erol, 1 mM EDTA, and 10 mM sodium sulfite. The enzymewas eluted in 2-ml fractions with a descending linear gradientof Polybuffer 96 hydrochloride (11-fold dilution, pH 7.0),from pH 9 to 7.

Purification of GST from I. orientalis. All operations werecarried out at 0 to 4° C, but Phenyl-Sepharose CL-4B columnchromatography was carried out at room temperature (15 to200C). In all column chromatographies, equilibration andelution were carried out with a buffer solution containing20% glycerol, 1 mM EDTA, and 10 mM sodium sulfite. GSTsolution was concentrated by ultrafiltration, if necessary,with a Labo cassette by using the UF membrane NMWL1,000 (Millipore Corp.).

Purification steps. (i) Preparation of cell extracts. Cellsharvested from 50 liters of culture (wet weight, 600 g) weresuspended in 2 liters of 0.05 M potassium phosphate buffer,pH 7.0, containing 1 mM EDTA and 10 mM sodium sulfite.The cells were disrupted with a Dyno-Mill (agitation rate,3,000 rpm with glass beads of 0.25 to 0.50 mm in diameter)(Willy A. Bachofen, Maschinenfabrik, Basel, Switzerland),and the supernatant solution was obtained by centrifugation.

(ii) Protamine sulfate treatment. A 2% protamine sulfatesolution was added to cell extracts up to 10% of the totalprotein, and the precipitate formed was removed by centrif-ugation. The supernatant solution was dialyzed for 48 hagainst three changes of 20 liters of 0.05 M potassiumphosphate buffer, pH 7.0, containing 1 mM EDTA andsodium sulfite.

(iii) DEAE-cellulose column chromatography. Glycerol wasadded to the dialyzed solution to make a 20% concentration.This enzyme solution was then applied to a DEAE-cellulosecolumn (10 by 50 cm) equilibrated with 0.05 M potassiumphosphate buffer, pH 7.0. The enzyme was eluted with thesame buffer. Active fractions were combined and concen-trated by ultrafiltration.

(iv) Sephadex G-100 gel filtration. The enzyme solutionwas divided into three portions, and each portion wasapplied to a Sephadex G-100 column (1.8 by 125 cm)equilibrated with 0.05 M potassium phosphate buffer, pH7.0, and eluted with the same buffer. The active fractionswere combined and concentrated by ultrafiltration. Theenzyme solution was dialyzed against the 0.05 M potassiumphosphate buffer, pH 7.0, containing 0.25 M NaCl.

(v) First Phenyl-Sepharose CL-4B column chromatography.The enzyme solution was divided into two portions, andeach portion was applied to a Phenyl-Sepharose CL-4Bcolumn (2 by 25 cm) equilibrated with 0.05 M potassiumphosphate buffer, pH 7.0, containing 0.25 M NaCl. After thecolumn was washed with the same buffer, the elution wascarried out with 0.05 M potassium phosphate buffer contain-ing 30% ethylene glycol. Two peaks of activity were eluted,and the fractions containing the activity were combined

separately for each peak. The second eluted peak was usedfor the study of enzymatic properties without further purifi-cation (GST Y-2).

(vi) CM-Sepharose CL-4B column chromatography. Thefirst eluted active peak from Phenyl-Sepharose CL-4B col-umn chromatography was applied to a CM-Sepharose col-umn (0.9 by 12 cm) equilibrated with 0.05 M potassiumphosphate buffer, pH 7.0. The enzyme activity was elutedwith the same buffer. The active fractions were combinedand dialyzed against 0.05 M potassium phosphate buffer, pH7.0, containing 0.25 M NaCl.

(vii) Second Phenyl-Sepharose column chromatography.The enzyme solution was applied to a Phenyl-Sepharosecolumn (1.4 by 43 cm) equilibrated with 0.05 M potassiumphosphate buffer, pH 7.0, containing 0.25 M NaCl. After thecolumn was washed with the same buffer, the enzyme waseluted with 0.05 M potassium phosphate buffer, pH 7.0,containing 0.25 M NaCl and 25% ethylene glycol. The activefractions were combined and used for the study of enzymaticproperties (GST Y-1).

Preparation of antisera. Antiserum was raised against GSTY-2 in white rabbits. GST Y-2 obtained by the first Phenyl-Sepharose CL-4B column chromatography was subjected toSDS-PAGE for further purification. After staining, the bandof GST Y-2 was cut out and eluted electrophoretically.About 1 mg of eluted protein was dialyzed thoroughlyagainst distilled water and freeze-dried. This preparationgave a single band on SDS-PAGE. Prepared GST Y-2antigen was dissolved in phosphate-buffered saline (12.5 mMsodium phosphate [pH 7.4], 150 mM NaCl), emulsified withan equal volume of Freund complete adjuvant, and injectedintramuscularly into male rabbits on days 1 and 22. Therabbits were bled from an ear vein 7 days after the secondinjection. The serum, containing 0.1% NaN3, was stored at4°C. A stained SDS-PAGE gel with no protein was alsoeluted, and the eluate was injected into the rabbits in thesame way. The serum obtained did not form precipitin linesagainst GST Y-1, GST Y-2, cell extract, or control serawhich were obtained from rabbits before immunization.

Immunodiffusion. The Ouchterlony immunoprecipitintests (24) were performed in 1% purified agar gel (Difco)containing 0.1% NaN3 and 0.05 M Tris hydrochloride, pH8.0.

RESULTS

Enzyme purification. The purification of GST was carriedout from the cell extract of I. orientalis by protaminetreatment and five column-chromatographic fractionations(Table 1). With the first Phenyl-Sepharose column chroma-tography, two peaks having GST activity were eluted (Fig. 1)and were named fraction A and fraction B. Fraction B gavea single band on disc PAGE (Fig. 2A, lane b), but fraction Agave several bands, including the same band as fraction B.Fraction B was designated GST Y-2 and characterized, andfraction A was further purified. Disc PAGE of fraction Aafter CM-Sepharose CL-4B column chromatography gavetwo protein bands (Fig. 2A, lane a), and each band extractedfrom the gel before staining showed GST activity.The heavy band showed the same migration with GST Y-2

on SDS-PAGE. At the final step, the second Phenyl-Sepha-rose column chromatography, the active fraction was elutedby 0.05 M potassium phosphate buffer, pH 7.0, containing25% ethylene glycol and 0.25 M NaCl. It gave only the lightprotein band on disc PAGE (Fig. 2A, lane c) and was namedGST Y-1.

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TABLE 1. Summary of purification

Step Purification procedure Total protein (mg) Total activity (mU) Sp act (mU/mg) Yield (%)

Cell extract 38,400 239,000 6.2 100ii Protamine 16,400 361,000 22.0 151iii DEAE-cellulose 3,950 130,000 32.9 54.4iv Sephadex G-100 130 79,200 609 33.1V First Phenyl-Sepharose CL-4B

Fraction A" 22.8 42,000 1,840 17.6Fraction B (GSTY-2) 2.3 1,400 609 0.59

Vi CM-Sepharose CL-4B 14.2 36,300 2,560 15.2Vii Second Phenyl-Sepharose CL-4B (GST Y-1) 0.09 570 6,330 0.24

" Only fraction A was submitted to steps vi and vii.

25% ethylene glycol and 0.25 M NaCl. It gave only the lightprotein band on disc PAGE (Fig. 2A, lane c) and was namedGST Y-1.

Molecular properties of GST Y-1 and GST Y-2. The mo-

lecular weights of GST Y-1 and GST Y-2 were determined tobe ca. 37,500 and 40,000 by gel filtration with high-perfor-mance liquid chromatography. SDS-PAGE of GST Y-1 andGST Y-2 gave single bands of 22,000 and 23,500, respec-

tively (Fig. 2B), indicating that each isoenzyme is a ho-modimer. Isoelectric points of GST Y-1 and GST Y-2 were

determined to be pH 8.40 and 8.55, respectively, from theresults of chromatofocusing.

Immunological properties. To demonstrate the immunolog-ical characteristics of GST Y-1 and GST Y-2, immunodiffu-sion experiments were performed. The antiserum to GSTY-2 reacted with two kinds of cell extract of I. orientaliscultured with or without o-DNB, and both formed a precip-itin line that showed identity with that of purified GST Y-2(Fig. 3). On the other hand, the antiserum to GST Y-2 did notreact with purified GST Y-1 (Fig. 3). These results suggestthat GST Y-1 is immunologically distinguishable from GSTY-2.

Catalytic properties. The properties of the purified isoen-zymes were investigated with CDNB as an electrophilicsubstrate. The optimum pH values for the conjugation ofGSH with CDNB by GST Y-1 and GST Y-2 were 7.0 and 7.0to 7.5, respectively. The optimum temperatures for theconjugation of GSH with CDNB by GST Y-1 and GST Y-2

were 450C and 350C, respectively. The pH stabilities of GSTY-1 and GST Y-2 were investigated by preservation of theseisoenzymes in various buffers containing stabilizing agents at40C for 1 week. GST Y-1 retained more than 90% activity inthe pH range of 6.5 to 7.5, but lost ca. 40% of its activitybelow pH 5.0 and above 9.0. GST Y-2 retained more than85% activity in the pH range of 6.5 to 11.7, but lost 60% ofits activity at pH 3.8. The temperature stabilities of theseisoenzymes were investigated by incubation of the isoen-zymes at various temperatures. GST Y-1 retained 92% of theinitial activity on incubation at 40'C for 10 min and lost 96%of the activity on incubation at 70'C. GST Y-2 retained 95%of the initial activity on incubation at 40'C for 10 min andretained 34% of the activity after boiling for 10 min.

Substrate specificities. The conjugation of GSH with vari-ous electrophilic substrates was measured spectrophotomet-rically (Table 2). GST Y-1 showed higher specific activity(6.33 U/mg of protein) than GST Y-2 (0.61 U/mg of protein)when CDNB was used as an electrophilic substrate. Wheno-DNB was used as the substrate, GST Y-1 also showedhigher specific activity (0.49 U/mg of protein) than GST Y-2(0.08 U/mg of protein), but when other substrates such as

bromosulfophthalein and trans-4-phenyl-3-buten-2-one were

used, GST activity was not detected for either isoenzyme.K,,, values of GST Y-1 for GSH and CDNB were 0.51 mM

cu -

cl) (Q)C

0.3

Ec-

ToCX 0.2iC\J0

a)u 0.1C

0

0U)-0o

100 200 300

Q)

-Q

U)2.0 C

D

0

0 C)(.

Fraction number (5ml/tube)FIG. 1. First Phenyl-Sepharose CL-4B column chromatography

of . orientalis GST. Approximately 30 U of enzyme was applied on

a Phenyl-Sepharose column, and chromatography was performed as

described in Materials and Methods. Symbols: 0, GST activity; *,A280.

(0) ( b) (c)

_m- P GST Y-2GST Y-I

MW

94 K

67 K

43 K

30 K

4~ 20.1 K

_ 14.4 K

(A) DISC-PAGE (B) SDS-PAGEFIG. 2. PAGE of I. orientalis GST Y-1 and GST Y-2. (A) Disc

PAGE. Electrophoresis and staining were carried out as describedin Materials and Methods. Lanes: a, 40 [Lg of active fraction from

CM-Sepharose; b, 20 jig of fraction B (GST Y-2); c, 10 pug of active

fraction eluted from the second Phenyl-Sepharose column (GSTY-1). (B) SDS-PAGE. Approximately 15 ,ug of GST Y-1 and GST

Y-2 were applied.

Frac.A Frac. B- -

30%Ethylene glycol

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FIG. 3. Immunodiffusion of GST Y-1 and GST Y-2 with antise-rum to GST Y-2 Wells: 1, homogeneous GST Y-2 (0.13 mg ofprotein per ml); 2, cell extract of f. orientalis cultured with o-DNB;3, cell extract of I. orientalis cultured without o-DNB; 4 through 6,homogeneous GST Y-1 (wells 4, 5, and 6 contained 0.29, 0.09, and0.02 mg of protein per ml, respectively); center well, GST Y-2antiserum.

presence of various GST inhibitors with CDNB as an elec-trophilic substrate (Table 3). Both GST Y-1 and GST Y-2were inhibited by hematin rather than tributyltin acetate andtriphenyltin chloride. Cibacron blue 3G-A inhibited GST Y-1significantly, whereas GST Y-2 was strongly inhibited bybromosulfophthalein.

DISCUSSIONGlutathione transferase activity is widely distributed, from

mammals to microorganisms (14). Especially in mammals,many tissue-specific (11, 12, 16) and species-specific (2, 3, 9,20) GST isoenzymes have been purified, and their propertiesand functions in cells have been studied extensively. GSTactivities have also been found in plants (10) and insects (8),and they are related to resistance to herbicides and insecti-cides. For microorganisms, there are only a few reportsabout GST activity (15, 18, 23). GST was purified andcharacterized only from Mucorjavanicus (1); it has not beenpurified from bacteria or yeasts. Recently, we reported that

TABLE 2. Substrate specificities of GST Y-1 and GST Y-2

Sp act in mU/mgaSubstrate

GST Y-1 GST Y-2

1-Chloro-2,4-dinitrobenzene 6,300 (100) 609 (100)o-Dinitrobenzene 490 (7.7) 81 (13.3)1,2-Dichloro-4-nitrobenzene ND NDBromosulfophthalein ND NDp-Nitrobenzoyl chloride ND ND1,2-Epoxy-3-(p-nitrophenoxy)-propane ND NDEthacrynic acid ND NDp-Nitrophenethyl bromide ND NDtrans-4-Phenyl-3-buten-2-one ND ND4-Nitropyridine-N-oxide ND ND

a GST activity was measured by a spectrophotometric method as describedin Materials and Methods. Relative rates are shown in parentheses. ND, Notdetectable under assay conditions used.

TABLE 3. Inhibition of GST Y-1 and GST Y-2

150 value (p.M)aInhibitor

GST Y-1 GST Y-2

Cibacron blue 3G-A 0.9 >40Tributyltin acetate >50 40Triphenyltin chloride 17 37Bromosulfophthalein 27 3.2Hematin 3.6 5

II50 value is the concentration of inhibitor giving 50% inhibition of GSTactivity. CDNB was used as a substrate.

6ST activities were widely distributed in yeasts and wereinduced by o-DNB, one of the substrates of GST in I.orientalis (16a). Thus, we used a culture medium containing200 p.M of o-DNB for purification of GST in this study.GST of I. orientalis is very unstable, and it loses almost all

of its activity by ammonium sulfate precipitation or bystanding at 40C for 1 week without stabilizing agents (16a).During purification, we could not use ammonium sulfatefractionation, and all operations had to be carried out in thepresence of stabilizing agents in the pH range of 6.5 to 7.5.We also could not use an affinity gel such as S-hexylglu-tathione-agarose because both isoenzymes did not adsorb tothe affinity column. Under these very restricted conditions,two GST isoenzymes were isolated to homogeneity for thefirst time from I. orientalis. The yield of GST Y-1 was lowbecause it was isolated by a slight difference of hydropho-bicity between GST Y-1 and GST Y-2. When a gradient wasused in Phenyl-Sepharose column chromatography, GSTactivity was eluted gradually and no separation of GST Y-1was observed.

Multiple forms of GST in an organism is a prominentfeature in the occurrence of the enzyme (22), and we haveobtained two isoenzymes, GST Y-1 and GST Y-2 from I.orientalis. Both GST isoenzymes are homodimers, contain-ing subunits with molecular weights of 22,000 (GST Y-1) and23,500 (GST Y-2).Antiserum to GST Y-2 reacted with two kinds of cell

extracts of I. orientalis, and each formed a single line ofprecipitation that showed identity with that of homogeneousGST Y-2. On the contrary, antiserum to GST Y-2 did notreact with homogeneous GST Y-1. From these results, twokinds of GST were distinguishable from each other; it is veryinteresting to study the role and relation of the two isoen-zymes in the cell.

Molecular properties of GST Y-1 and GST Y-2 are similarto GSTs from mammals (22), but hybridization of subunits,which underlies the occurrence of multiple isoenzymes in amammal, was not observed in our isoenzymes. Isoelectricpoints of the isoenzymes, pH 8.40 for GST Y-1 and pH 8.55for GST Y-2, correspond to those of basic GSTs of mam-mals. Both isoenzymes showed high substrate specificitytoward CDNB, as was reported for mammalian and M.javanicus (1) GSTs. GST Y-1 showed higher specific activ-ities than GST Y-2 when o-DNB or CDNB was used as asubstrate. Although o-DNB was used as an inducer of GST,the relative rates of GST reaction toward o-DNB were lowcompared with those toward CDNB. Relation of the sort ofinducer and the molecular species of GST synthesized in theyeast cells is interesting and is a problem to be elucidated.GSH peroxidase activity, which has been reported to coexistin some mammalian GST (19), was not detected in ourisoenzymes.

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4. Asaoka, K., and K. Takahashi. 1983. A colorimetric assay ofglutathione S-transferases using o-dinitrobenzene as a sub-strate. J. Biochem. 94:1685-1688.

5. Bach, M. K., J. R. Brashler, D. R. Morton, L. K. Steel, M. A.Kaliner, and T. E. Hugli. 1982. Formation of leukotrienes C andD and pharmacologic modulation of their synthesis. Adv. Pros-taglandin Thromboxane Leukotriene Res. 9:103-114.

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7. Boyland, E., and L. F. Chasseaud. 1969. The role of glutathioneand glutathione S-transferases in mercapturic acid biosynthesis.Adv. Enzymol. 32:173-219.

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10. Guddewar, M. B., and W. C. Dauterman. 1979. Purification andproperties of a glutathione S-transferase from corn which con-jugates S-triazine herbicides. Phytochemistry (Oxf.) 18:735-740.

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16. Jensson, H., P. Alin, and B. Mannervik. 1985. Glutathionetransferase isoenzymes from rat liver cytosol. Methods En-zymol. 113:504-507.

16a.Kumagi, 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.

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18. Lau, E. P., L. Niswander, D. Watson, and R. R. Fall. 1980.Glutathione S-transferase is present in a variety of microorgan-isms. Chemosphere 9:565-569.

19. Lawrence, R. A., and R. F. Burk. 1976. Glutathione peroxidaseactivity in selenium-deficient rat liver. Biochem. Biophys. Res.Commun. 71:952-958.

20. Lee, C.-Y., L. Johnson, R. H. Cox, J. D. McKinney, and S.-M.Lee. 1981. Mouse liver glutathione S-transferases. J. Biol.Chem. 256:8110-8116.

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22. Mannervik, B. 1985. The isoenzymes of glutathione transferase.Adv. Enzymol. 57:357-417.

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