THE JOURNAL OP Broma~cn~ CHEMISTRY Vol. 249, No. 22, Issue of November 25, PP. 7130-7139, 1974

Print& in U.S.A.

Glutathione S-Transferases

THE FIRST ENZYMATIC STEP IN MERCAPTURIC ACID FORMATION*

(Received for publication, May 1, 1974)

WILLIAM H. HABIG, MICHAEL J. PABST,~ AND WILLIAM B. JAKOBY

From the Section on Enzymes and Cellular Biochemistry, National Institute of Arthritis, Metabolism and Diges- tive Diseases, National Institutes of Health, Bethesda, Maryland 20014

SUMMARY

The purification of homogeneous glutathione S-transfer- ases B and C from rat liver is described. Kinetic and physi- cal properties of these enzymes are compared with those of homogeneous transferases A and E. The letter designations for the transferases are based on the reverse order of elution from carboxymethylcellulose, the purification step in which the transferases are separated from each other. Transfer- ase B was purified on the basis of its ability to conjugate iodo- methane with glutathione, whereas transferase C was puri- fied on the basis of conjugation with 1,2-dichloro-4-nitro- benzene. Although each of the four enzymes can be identi- fied by its reactivity with specific substrates, all of the en- zymes are active to differing degrees in the conjugation of glutathione with p-nitrobenzyl chloride. Assay conditions for a variety of substrates are included.

All four glutathione transferases have a molecular weight of 45,000 and are dissociable into subunits of approximately 25,000 daltons. Despite the similar physical properties and overlapping substrate specificities of these enzymes, only transferases A and C are immunologically related.

Glutathione S-transferases (EC 2.5.1.18) are thought to play a physiological role in initiating the detoxication of poten- tial alkylating agents (l-3)) including pharmacologically active compounds. These enzymes catalyze the reaction of such com- pounds (Fig. 1) with the -SH group of glutathione, thereby neutralizing their electrophilic sites and rendering the products more water-soluble. Glutathione conjugates are thought to be metabolized further by cleavage of the glutamate and glycine residues, followed by acetylation of the resultant free amino group of the cysteinyl residue, to produce the final product, a mercapturic acid (2, 3). The mercapturic acids, i.e. S-alkylated derivatives of N-acetylcysteine, are then excreted.

There is a voluminous amount of literature which deals with individual glutathione transferase reactions in relatively crude enzyme preparations from rat liver. Attempts have been made

* This work is dedicated to Otto Hoffmann-Ostenhof on the occasion of his 60th birthday.

$ Present address, School of Dentistry, University of Colorado Medical Center, Denver, Colo.

to classify such enzyme activities on the basis of the carbon skeleton of the electrophilic molecule or the specific leaving group involved (2), hence the formerly common use of the terms aryl- (4), alkyl- (5), alkene- (6), and epoxidetransferase (7, 8). Our findings are not in accord with such designations, since each of the six glutathione transferases which we have purified has overlapping specificities (9, 10). In order to avoid ambiguity we have chosen to assign letters to the transferases, based upon their order of elution from CM-cellulose, a step in the purifica- tion method common to each of the enzymes described.

This report outlines procedures for obtaining homogeneous preparations of two of the enzyme species, glutathione trans- ferases II and C, and compares homogeneous transferases A, B, C, and E in terms of their physical properties and substrate specificity. The purification and a kinetic analysis of the mech- anism of transferase A are described separately (10) ; the prep- aration of transferase E and its activity with epoxides have been reported previously (8).

EXPERIMENTAL PROCEDURE

Materials and Methods

Chemicals-1,2-Dichloro-4-nitrobenzene, 4-nitropyridine-N-ox- ide, and p-nitrobenzyl chloride were obtained from Aldrich Chemical Co. and were recrystallized from ethanol-water before use. The sodium salt of bromosulfophthalein was obtained from Sigma Chemical Co. and 1-menaphthyl sulfate from Calbiochem. Donald Jerina of this Institute provided 1,2-naphthylene oxide (ll), P. R. Carnegie of the University of Melbourne provided homoglutathione (L-y-glutamyl-L-cysteinyl-fl-alanine) (12), and Horace D. Brown of Merck Sharpe and Dohme provided etha- crynic acid ([2,3-dichloro-4-(2-methylenebutyryl)phenoxy]acetic acid). The other compounds were readily available commercial products and were used without further purification.

Activity Units-A unit of activity is defined as the amount of enzyme catalyzing the formation of 1 rmole of product per min under the conditions of the specific assay. Specific activity is defined as the units of enzyme activity per mg of protein as meas- ured by the method of Lowry et al. (13), with bovine serum albu- min as a standard.

Spectrophotometric Assay Methods-Enzyme activity with aro- matic substrates was usually determined by monitoring changes in absorbance in a Cary 15 dual beam spectrophotometer. A complete assay mixture without enzyme was used as a control. Assays were conducted in a thermostated cell compartment at 25” in 0.1 M potassium phosphate at a pH at which the nonenzy- matic reaction was minimal (Table I). The concentration of GSH was 5 mM, except in the systems with trans.4-phenyl-3-bu- ten-2-one and ethacrynic acid (0.25 mM GSH) and with l-chloro- 2,4-dinitrobenzene (1 mM GSH). The concentration of the

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8 9 10

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Br 0

k03Na

SO,Na

FIG. 1. Substrates for glutathione transferases: 1, 1,2-di- 8, 1-menaphthyl sulfate; 9, trams-4-phenyl-3-buten-2-one; 10, chloro&nitrobenzene; d, 1-chloro-2,4-dinitrobenzene; 3, p-nitro- p-nitrophenethyl bromide; If, bromosulfophthalein. Leaving benzyl chloride; 4,4-nitropyridine-N-oxide; 5,1,2-epoxy-3-(p-ni- groups are circled, whereas arrows indicate the site of addition of trophenoxy)propane; 6, 1,2-naphthalene oxide; 7, iodomethane; GSH to alkenes and epoxides.

TABLE I

Conditions for specttophotometric enzyme assays in 0.1 M potassium phosphate and 6 my GSH at 86’

Substrate [Substrate]

1,2-Dichloro-4-nitrobenzenea

1-Chloro-2,4-dinitrobenzenel

4-Nitropyridine-N-oxide

p-Nitrophenethyl bromide

p-Nitrobenzyl chloride

1,2-Epoxy-3-(p-nitrophenoxy)propane

1,2-Naphthalene oxide

Bromosulfophthalein

1-Menaphthyl sulfate5

trans-4-Phenyl-3-buten----oneA

Ethacrynic acidd

mM -

1.0

1.0

0.2

0.1

1.0

5.0

0.1

0.03

0.5

0.05

0.2

PH

7.5

6.5

7.0

6.5

6.5

6.5

8.5

7.5

7.5

6.5

6.5

A max

E!!

345

340

295

310

310

360

260

330

298

290

270

na

mM-lcm -1

8.5

9.6

7.0

1.2

1.9

0.5

8.1

4.5e

2.5

-24.8

5.0

a_ Modification of an assay devised by Booth et al. (1). --

b At a GSH concentration of 1 m&i.

c Assay devised by Gillham (14). d -At a GSH concentration of 0.25 m$

e Data derived frcan Fig. 7 of Goldstein and Combes (15).

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specific substrates listed in Table I was limited by either high absorbance or low solubility; in most cases, the substrate concen- tration was equal to or less than the K,. The wavelength used (Table I) was the maximum in a difference spectrum between the starting assay mixture and the assay mixture after complete enzymatic conversion to product. The difference in the molar extinction coefficient (Ae) was also obtained from such a deter- mination. All assays were linear functions of protein concen- tration and of time for at least 3 min when the amount of enzyme used resulted in an absorbance change of less than 0.05 per min. Substrates of limited water solubility were prepared in ethanol; the final ethanol concentration in the assay solution was always less than 4%.

Titrimetric Assay-The conjugation of alkyl halides with GSH was measured by a variation of the titrimetric procedure of Johnson (5). The rate of addition of 5 mM NaOH required to maintain the pH at 7.2 was measured in the stirred reaction vessel of a Radiometer type TTTlc recording titrimeter. The standard assay mixture consisted of 3.25 ml of 9.5 mM iodomethane and 1.5 mM neutralized GSH. The nonenzymatic rate was measured for several minutes prior to the addition of 10 to 50 ~1 of the protein sample. Full scale on the recorder represented the addition of 2.5 pmoles of base. Typically, less than 1 rmole of substrate was utilized during each assay. Enzyme activity was linear in the range of 0.01 to 0.4 pmole of product formed per min. Samples of low activity and high buffering capacity, e.g. fractions from hydroxylapatite columns, required dialysis prior to assay. This was conveniently performed by ultrafiltration through a PM-10 filter followed by dilution with unbuffered solvent.

Nitrite Assay-Nitrite was determined by the method of Snell and Snell (16). Reaction mixtures consisted of 2.0 ml of 0.1 M

potassium phosphate, pH 7.5, containing the nitro compound as substrate and 0.2 ml of 50 mM GSH. After addition of enzyme, aliquots of 0.4 ml were removed at intervals, usually at 0, 5, 15, 30, and 60 min, and added directly to 2.0 ml of 1% (w/v) sulfanil- amide in 2vo HCl. To this mixture were added 2.0 ml of 0.02% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride, and the reaction vessel was briefly shaken. After 20 min, color develop- ment was measured in a Klett-Summerson calorimeter with a 540 nm filter. One micromole of nitrite was equivalent to approxi- mately 5000 Klett units. Assays, corrected for nonenzymatic nitrite release, were linear in the range of 0.1 to 10 nmoles of nitrite formed per min.

GSH Assay-The conjugation of ally1 alcohol (2-propen-l-01) with glutathione was measured by the disappearance of free sulfhydryl groups using 5,5’-dithiobis(2-nitrobenzoate) (17). In addition to enzyme, the assay mixture included 0.1 M potassium phosphate (pH 6.5), 10 mM ally1 alcohol, and 1 mM GSH. Enzyme activity was linear in the range of 0.1 to 10 nmoles of GSH con- sumed per min per ml.

Sodium Dodecyl Sulfate-Gel Electrophoresis-Electrophoresis in sodium dodecyl sulfate gels was performed as described by Weber and Osborne (18). Calibration proteins and their molecular weights included lysozyme (14,300)) myoglobin (17,200)) trypsin (23,500), carbonic anhydrase (29,000), carboxypeptidase (34,500), aldolase (40,000), and glutamate dehydrogenase (53,000).

Sedimentation Equilibrium Centrifugation-The equilibrium distribution of transferase B was measured at 22,000, 26,000, and 28,000 rpm in a Beckman model E analytical ultracentrifuge equipped with the photoelectric scanning system. Absorbance was measured at 280 nm in an An-G rotor at 6”. The enzyme was in a solution of 30yo glycerol, 5 mM GSH, 1 mM EDTA, and 0.1 M

potassium phosphate (pH 6.7), with a measured density of 1.1017 g per cm3 at 6”.

Gel Filtration-Samples were examined with a calibrated column (2.0 X 45 cm) of Sephadex G-75 in 0.1 M potassium phosphate, pH 6.7. Standards included pancreatic ribonuclease (23,700), chicken ovalbumin (45,000), and bovine serum albumin (68,000).

Amino Acid Analysis-Homogeneous enzyme, 1.5 mg in 1 ml, was dialyzed overnight against 2 liters of 10 mM potassium phos- phate, pH 6.7, containing 1 mM dithiothreitol. Dialysis was continued for an additional 3 days against changes of buffer without the mercaptan. The sample was divided into aliquots for 24, 48, and 72 hours of hydrolysis. Analyses were performed with a Beckman model 120C amino acid analyzer. Methionine and cysteine were determined after treatment with performic

0 0 30 60 so 120 150 100 210

FRACTION NUMBER

FIG. 2. Elution pattern of protein and glutathione transferases from CM-cellulose column. 0, activity with 1,2-dichloro-4-ni- trobenzene; A, activity with iodomethane; 0, activity with 1,2-epoxy-3-(p-nitrophenoxy)propane; . . . . , absorbance at 280 nm; -, the conductivity.

acid, as described by Hirs (19). Tryptophan was determined spectrophotometrically (20).

Antibody Production-Three young, male New Zealand rabbits (1 kg) each received injections of a total of 1.5 mg of transferase A over a period of 35 days. Initial injections contained antigen with Freund’s adjuvant and were administered at multiple intra- muscular sites. Multiple subcutaneous injections of transferase A in buffered saline were used to boost the immune response. Animals were bled by cardiac puncture 10 days after final injec- tion. Y-Globulin fractions of the rabbit antisera were prepared by the ammonium sulfate procedure of Hebert et al. (21).

Preparation of Enzymes

Livers from male Sprague-Dawley rats (Gibco Microbiological Laboratories) were obtained under the conditions described previously (8) and stored at -80” until required. The livers, 500 g from 50 rats, were pulverized while frozen and then allowed to thaw partially in 1.6 liters of water. All subsequent steps were performed at 4’. The tissue was homogenized in a Waring Blendor for 1 min and centrifuged for 1.5 hours at 10,000 X g. The supernatant fluid was filtered through a plug of glass wool to remove floating lipid.

The extract was passed through a column of DEAE-cellulose (Whatman DE-52) (15 cm in diameter X 20 cm high) equilibrated with 10 mM Tris-chloride, pH 8.0. The column retained about 80% of the applied protein, whereas enzyme activity, as assayed with 1,2-dichloro-4-nitrobenzene, was not adsorbed.’ The col-

umn was rinsed with the same buffer until no further activity emerged. Ammonium sulfate, 660 g per liter, was added to the active eluate and the preparation was centrifuged at 10,000 X g for 30 min. The precipitate was dissolved in 150 ml of 10 mM potassium phosphate, pH 6.7 (Buffer A), and was dialyzed for 1 day against three changes of a total of 8 liters of Buffer A.

The dialyzed preparation was applied to a column of CM-cel- lulose (Whatman CM-52) (5 X 40 cm) equilibrated with Buffer A. After rinsing with 2 liters of the same buffer, a 4-liter linear salt gradient composed of equal amounts of Buffer A and Buffer A containing 75 mM KC1 was applied to the column. Fractions of 15 ml were collected.

The four peaks of transferase activity eluted from CM-cellulose (Fig. 2) were designated alphabetically, with A representing the species eluted at the highest ionic strength. The subsequent purification and kinetic properties of transferase A are presented separately (10). The peak of transferase activity labeled D&E is known to be composed of at least two distinct enzyme species: transferase E has been purified to homogeneity and has been

1 One glutathione transferase, transferase M, which is adsorbed by DEAE-cellulose (9), is active with menaphthyl sulfate as the electrophile and has very low activity with 1,2-dichloro-4-nitro- benzene.

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TABLE II Summary of purification of transferases B and C

Purification step

1

2

3

4B

4c

5B

5c

6~

6C

Crude extract

DEAE-Cellulose

Ammonium sulfate

CM-Cellulose

CM-Cellulose

Hydroxylapatite 1

Hydroxylapat ite 1

Hydroxylapatite 2

Hydroxylapatite 2

Volume

ml -

1400

1520

190

35

21

38

12

17

15

-- Tota 1

protein

%

61,000

8,700

7,100

700

570

98

56

68

48

I

Total activity Specific activity

I.lmole

360

665

665

140

min -1

1100

940

860

pmoles I

0.006

0.08

0.09

0.20

230 0.40

65 0.66

100 1.8

55 0.81

-1 -1 I mg

0.018

0.11

0.12

96 2.0

L

a Determined by standard titrimetric assay with iodometbane as substrate.

b Determined by standard spectrophotometric assay with 1,2-dichloro-4-

-nitrobenzene as substrate.

implicated in the conjugation of GSH with epoxides (8) and p-nitrophenethyl bromide (9), whereas transferase D is active with epoxides but not with p-nitrophenethyl bromide. Trans- ferase D has not yet been purified extensively.

Further Purijkation of TTansfeTase B-Fractions 156 through 176 from the CM-cellulose column were pooled and concentrated to 35 ml by ultrafiltration through a Diaflo PM-10 membrane. The concentrated solution, dialyzed against 1 liter of 10 mM potassium phosphate (pH 6.7) containing 30% glycerol, 2 mM GSH, and 0.1 mM EDTA (Buffer B), was applied to a column of hydroxyl- apatite (2.0 X 20 cm) equilibrated with Buffer B. After washing with 200 ml of Buffer B, the column was developed with a 600-ml linear gradient of 10 to 400 mM potassium phosphate (pH 6.7) in asolution containing 300/O glycerol, 2 mM GSH, and 0.1 mM EDTA. Fractions of 10 ml each were collected and numbered, beginning with the sample application. Three activity peaks, each capable of conjugating 1,2-dichloro-4-nitrobeneene with glutathione, were located at Fractions 33, 41, and 49. The first group, Frac- tions 31 to 36, represented only 2oj, of the 1,2-dichloro-4-nitro- benzene-conjugating activity of the eluate but was coincident with all of the iodomethane-conjugating activity.

Fractions 31 to 36 were concentrated to 10 ml, dialyzed against Buffer B overnight, and applied to a slightly smaller column (2.0 X 16 cm) of hydroxylapatite; the column was developed as in the previous step. A single peak of protein w&s found in Fractions 23 through 27; coincident with this peak were the iodomethane- and weak 1,2-dichloro-4-nitrobenzene-conjugating activities. Fractions 24 and 25 were pooled, dialyzed against Buffer B, and stored at -80” without concentration.

Table II presents a summary of the purification procedure for transferase B. A final yield of 8% of the iodomethane-conju- gating activity of the initial extract was obtained. However, since Fractions 156 through 176 from the CM-cellulose column contained only one-half of the iodomethane-conjugating activity eluted from the column (the other 50% residing with transferase E), the actual recovery is estimated as 16% of the original trans- ferase B activity.

Further Purification of Transferase C-Fractions 133 through 143 from the CM-cellulose column were pooled for the purification

+ A

E

FIG. 3. Gel isoelectric focusing of transferases A, B, and C. Approximately 50 pg of each transfer-e were examined in acryl- amide gels containing pH 7 to 10 ampholytes. The isoelectric points of the activity peak of transferases A and B, determined by preparative electrofocusing in a sucrose density gradient, are pH 8.9 and pH 9.8, respectively.

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TABLE III Ami?Lo acid analysis

Moles of amino acid/45,000 g transferase’

Lys ine

Histidine

Arginine

Aspartic acid

Threoninell

Ser inch

Glutamic acid

Proline

Glycine

Alanine

CysteineC

Va 1 ine

Methionine’

Is 0 leuc ine

Leucine

Tyr os ine

Phenylalanine

TryptophaG

Transferase A Transferase B Trans ferase C

34 36 35

6 6 6

21 22 21

45 37 44

13 11 12

20 18 17

42 46 41

22 20 22

19 21 21

20 31 20

6 4 6

11 25 12

10 8 10

22 18 19

45 50 45

23 13 22

20 17 22

6 9 6

a The actual molecular weights calculated from the amino acid values

presented here are 45,202, 45,053, and 44,728 for transferases A,

B and C, respectively.

?L Estimated by extrapolation to zero hydrolysis time.

c Determined after performic acid oxidation of sample (19) o a Determined by the method of Edelhoch et al. (20).

of transferase C. The buffers, column sizes, and procedures used were identical with those described for the purification of trans- ferase A (10). Transferase C was eluted from the first hydroxyl- apatite column in Fractions 53 through 65, slightly before a small amount of contaminating transferase A in Fractions 71 through 78. A single peak of activity and protein was eluted following application of Fractions 53 through 65 to a second hydroxylapatite column which was developed in the same manner. A summary of purification of transferase C is also shown in Table II. The final yield was 9% of the 1,2-dichloro-4-nitrobenzene-conjugating activity of the original extract. The yield should be considered with the fact that less than 50% of the dichloronitrobenzene- conjugating activity is found in the transferase C peak of the CM-cellulose column.

RESULTS

Properlies of Enzymes

Homogeneity and di olecular Weight-Transferases 13 and C were found to be homogeneous by the criterion of sodium dodecyl sulfate-gel electrophoresis; a single band with a molecular weight of about 25,000 was found for each enzyme. In mixing experi- ments, the subunits of transferase A and transferase C migrated together and appeared to be identical by this criterion. Trans- ferase U was also examined by sedimentation equilibrium anal- ysis; a straight line was obtained when the log of protein con-

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centration was plotted as a function of the square of the distance C eIuted as single symmetrical peaks from Sephadex G-75; in from the center of the rotor. each case the elution position of the enzyme was identical with

Sedimentation equilibrium analysis at two concentrations of that of ovalbumin (45,000 daltons; 2?, 0.75). transferase 13, 50 and 100 ,ug per ml, yielded a molecular weight The enzymes were also examined by gel clectrofocusing (23) of 47,000, based on a partial specific volume (8) of 0.745 cm3 per in the range of pH 7 to 10. As reported for transferases A (10) g as calculated from the amino acid composition (22). Neither and E (S), each of the purified enzymes yields at least two bands aggregation nor dissociation was detected. Transferases 1) and of active enzyme. Two bands were also observed for trans-

.., : ““!

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'. ' iA . I /

..' ". ::..:-:' .:.: , 1 ', ,,.' .".,,> ,.,,,, ,-.,, ,..a f.,,,.( .:; .,.. .,,..: ,,.,; ,,., ,:,:;, ,:: /

A+C

FIG. 4. Immunoelectrophoretic analysis of transferases A and C. The concentration of each transferase was 0.8 mg per ml. Rabbit antiserum to transferase A was placed in the troughs after electrophoresis for 45 min at 6 volts per cm in 0.1 M sodium barbital (pH 8.6)-2oj, agar. Arrows indicate the point ot peak antigen concentration. The presence of a continuous line for the mixture of transferases A and C indicates that antibodies to trans- ferase A recognize similar antigenic determinants on both trans- ferases A and C.

PH PH

FIG. 5. The effect of pH on conjugation by glutathione trans- ferases. A, rates determined with iodomethane by the titrimetric assay. 0, nonenzymatic rate; 0, enzyme-catalyzed rate with 80 pg of transferase B; A, enzyme-catalyzed rate with 11 rg of transferase E. B, rates determined by spectral assay of trans-4- phenyl-3-buten-2-one conjugation with GSH. 0, nonenzymatic rate; 0, enzyme-catalyzed rate with 8 rg of transferase A; A, enzyme-catalyzed rate with 1.6 fig of transferase C. Potassium phosphate (0.1 M) was used at pH 6.5 and 7.0 and Tris-chloride (0.1 M) at higher pH values.

TABLE IV

Specific activities of the glutathione tramferases with selected substrates0

Substrate A B

1,2-Dichloro-4-nitrobenzene 4.3 0.003

l-Chloro-2,4-dinitrobenzene 62 11

p-Nitrobenzyl chloride 11.4 0.1

1,2-Epoxy-3-(p-nitrophenoxy)propane 0.1 < o.oo&

Naphthylene oxide' 0.04 0

Bromosulfophthalein 0.53 0.006

trans-4-Phenyl-3-buten-2-one 0.02 0.001

Ally1 alcohol 0.002 0.003

Iodomethane < 0.02s 0.59

Ethacrynic acid < 0.0s 0.26

Transferase

T C

2.0

10

10.2

<O.lC

0.18

0.40

0.003

< 0.01s

0.11

E

<0.0001~

0.01

4.1

6.7

0.16

0.0001~

< 0.0001~

0.008

8.9

< 0.01s

a Specific activities determined under standard assay conditions as

described in the text.

a Data obtained by Dr. Taro Hayakawa of the Roche Institute of

Molecular Biology.

c Although no enzyme activity was observed, this value represents the

lower limit of the assay at the highest enzyme concentration which

was used.

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ferases B and C (Fig. 3). Although attempts to elute active enzyme from either band of transferase B were ineffective, there is evidence for the existence of two forms of transferase B. After storage of this enzyme at the stage of the first hydroxyl- apatite step, chromatography on a second column of hydroxyl- apatite resulted in two peaks of activity in the ratio of 8:2. Both fractions had identical specific activities with respect to iodomethane and 1,2-dichloro-4-nitrobenzene. Chromatog- raphy of the major fraction on a third hydroxylapatite column again resulted in the same elution pattern, with the two active peaks appearing in a ratio of 9: 1. This formation of multiple forms was not affected by the addition of the serine protease inhibitor, phenylmethanesulfonylfluoride.

Amino Acid Analysis-The amino acid composition of trans- ferases A, B, and C are presented in Table III. The higher content of aromatic amino acids is reflected in the greater ab- sorbance of transferases A and C; ZZiz values are 12.5, 8.1, and 11.8 for transferases A, B, and C, respectively, based on chem- ical determination (13) of protein.

ImmunodQusion and Immunoelectrophoresis-Rabbit anti- body to rat liver transferase A reacted with both transferases A and C but not with transferases B or E when examined by Ouchterlony immunodiffusion (24). Immunoelectrophoretic analysis (25) indicated that transferases A and C share a com- mon antigenic determinant (Fig. 4) ; their electrophoretic mobil- ities are in accord with the isoelectric points of these molecules (Fig. 3). We are uncertain of the significance, if any, of the splitting of the arc given by transferase A alone (Fig. 4). Not shown are the results of immunodiffusion tests with a monovalent antibody preparation that reacted with transferase B and not at all with transferase A, C, or E.

Sulfhydryl Grcvups-The total numbers of free sulfhydryl

groups in 6 M guanidine hydrochloride for transferases A, B, and C were found to be 3.5, 4.2, and 3.5, respectively, when messure- ments were made with 5,5’-dithiobis(2-nitrobenzoate). Because of their immunological relatedness and their similarity in amino acid composition, transferases A and C were examined more closely with regard to their free -SW groups by following the rate of -SH titration. Whereas both enzymes have the same number of titrable -SH groups, the time required for reaction of three -SH groups with 0.67 mM 5,5’-dithiobis(2-nitrobenzo- ate) at pH 7.0 was 4 min for transferase A and 30 min for trans- ferase C.

pH Optima-The pH optimum for the conjugation of GSH with iodomethane by transferases B and E was 7.4 (Fig. 5A). The conjugation of trans-4-phenyl-3-buten-2-one with GSH was optimal at about pH 7.8 for transferase C and at pH 8.5 for transferase A (Fig. 5B). Thus, the optimal pH for conjugation varies among the tested substrates and among some of the trans- ferases acting on the same substrate. Transferase B has a pH optimum between 8.5 and 9.0 with 1,2-dichloro-4-nitrobenzene as substrate (not shown) ; this is similar to the broad optimum at pH 8.5 found with transferase A and this substrate (10).

Substrate Speci$city-Each of the glutathione transferases ob- tained in homogeneous form, transferases A, B, C, and E, were purified by assaying for activity with a specific electrophilic substrate. Thus, transferase E, which was isolated because of an interest in the conjugation of GSH with epoxides, was as- sayed in a system containing 1,2-epoxy-3-(p-nitrophenoxy)- propane as the electrophile (8). Indeed, the enzyme was re- ferred to as an epoxidase (26) and was listed as EC 4.4.1.7, S-(hydroxyalkyl)glutathione lyase (27), since its broader speci- ficity in catalyzing GSH conjugation was then unknown. Be- cause the literature (3) had suggested the presence of other

TABLE V Alkylhalides as transferase substrates

Specific activity as determined by titrimetric assay of 40 fig of the GSH concentration is 1.5 mM and that of the second substrate transferase A, 80 pg of transferase B, and 56 pg of transferase E, is5mM. The error values in the transferase assays in micromoles corrected for nonenzymatic activity. The nonenzymatic activity min-lmg-l are estimated as: transferase A, ~1~0.06; B, &0.03; and is not corrected for the effect of COZ absorption. In all cases, c, f0.04.

Substrate

Iod ome t ha ne

Iodoethane

l-Iodopropane

1-Bromopropane

1-Chloropropane

2 - Iodopropane

2-Bromopropane

2-Bromo-2-methylpropane

Nonenzyma t ic

activity

pmoles min -1

.019

.015

.009

.009

.009

.009

.Oll

.008

Specific activity

of transferase

(E A]B

E!E!

0

.Ol

.39

.08

.59 8.9

.17 2.8

.32 1.0

.09 .09

.03 0

.16 .63

.08 0

.08 0

min -1 -1

mg

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TABLE VI Nitroalkanes as transjerase substrates

Activities were determined from at least four assays over a period of 60 min. The GSH concentration was 5 mM in all experi- ments. The estimated uncertainties for specific activity of the transferases, in nanomoles min-img-i are: transferase A, fl.0; B, f0.4; C, ~0.5; and E, f1.4.

Substrate [Substrate]

Nitromethane

Nitroethane

l-Nitropropane

2-Nitropropane

2-Nitroethanol

3-Nitro-2-butanol

mM nmoles min -1 - --

20 .04

50 .17

20 .ll

50 .ll

20 .06

50 .07

20 .37

50 .53

20 .06

50 .12

20 .24

50 .66

Nonenzyma t ic

activity

Spec

of

.fic activity

transferase

AI B IC E

-1 -1 nmoles min mg

2.6 1.7 1.2

2.5 1.0 0.9

1.5 0.6 0.8

1.5 0.8 0.8

0.2 0.4 0.1

1.4 0.5 1.0

12.4 5.2 6.9

21.2 8.0 3.5

0 0.1 0

0.6 0.2 0.3

1.5 0.9 1.1

0 0 0.8

transferases with restricted specificity, enzymes were sought which would be active in the conjugation of GSH with iodo- methane, an “alkyl transferase” substrate (5), and with 1,2- dichloro-4-nitrobenzene, an “aryl transferase” substrate (4).

It is apparent from lable 1V that the enzymes do not fall into the clear pattern anticipated. Transferase B, for example, isolated by assay with iodomethane, has a lower activity with that substrate than does the enzyme, transferase E, which had been isolated by assay with an epoxide. In fact, transferase E has a higher specific activity with iodomethane than with the epoxide. However, under our assay conditions, transferase B represents the major component of iodomethane-conjugating activity in rat liver. The two enzymes could be differentiated with respect to this activity because transferase E was inhibited at concentrations of iodomethane greater than 10 mM; the in- hibition did not appear to be due to slow alkylation of the en- zyme, since the velocity was constant during the assay period. Transferase B showed no sign of inhibition at 50 mru iodomethane.

The effectiveness of several alkyl halides as substrates is shown in Table V. With transferase E, the rate of catalysis decreases with increasing chain length of the substrate, in agreement with the findings of an earlier study with a partially purified prepara- tion labeled as an “alkyl transferase” (5). This pattern was not reflected in the activity spectrum of transferase B. The enzy- matic reaction velocities of the propyl halides decrease in the expected order of I > Br > Cl. Although transferase A was

inactive with iodomethane, 1-iodopropane was a substrate for this enzyme.

The activity of a series of nitroalkanes is shown in Table IV. In this case, also, the reaction velocity decreased with increasing chain length for the primary nitro compounds. Transferase E was inactive with all of the tested compounds (Table VI), despite its high activity with the alkyl halides. The greater activities of transferases A, B, and C with 2-nitropropane are in contrast to their lower activities with the analogous substrates in the halide series.

Of the several aromatic compounds which were tested, l- chloro-2,4-dinitrobenzene, an “aryl transferase” substrate (4)) and p-nitrobenzyl chloride, an “aralkyl transferase” substrate (28), were active with all four enzymes, although there were dramatic differences in specific activity (Table 1V). /rans-4- Fhenyl-3-buten-a-one, an “alkene transferase” substrate (6), served for transferases A, B, and C. Bromosulfophthalein, a diagnostic reagent useful in studies of liver function (29) and known to undergo conjugation with GSH (30, 31), was a sub- strate for transferases A, B, and C; this compound was also a very effective substrate inhibitor of transferases A (10) and B. Ethacrynic acid, a compound of clinical importance as a diuretic (32), was most active with transferase B, although transferase C could also use it as a substrate.*

* No pH change accompanied the conjugation of this substrate, suggesting that the product results from addition of GSH to the methylene group rather than from displacement of a ring chloride.

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TABLE VII Kinetic parameters of the glutathione transfetases

Substrate

1,2-Dichloro-4-nitrobenzene

1-Chloro-2,4-dinitrobenzene

p-Nitrobenzyl chloride

Iodomethane

2-Nitropropane

trans-4-Phenyl-3-buten-2-oneb

Bromosulfophthalein

GlutathioneC

A

KmZ V max

1.1 390

0.06 3000

1.4 1200

0

29 2.2

0.3 25

.002 77

0.2

Transferase

B

K V m max -

2 1.3

0.8 860

3.4 60

40 200

9.2 0.5

.07 0.24

0.2

C

Km ‘max --

3.0 250

0.1 500

0.6 840

0

0.2 320

0.1

E

K V 2 max

0.8 430

0.7 560

0

0

0

2.0

CL Km is G, V is moles min-l/mole enzyme.

b Determined at nonsaturating GSH (0.25 I$ because of rapid nonenzymatic reaction.

c Determined with 1 m_M 3,4-dichloronitrobenzene

from reference (8) e

as substrate for enzymes A, B and C. Data for E

Naphthalene oxide, an arene oxide, was conjugated by trans- ferases A and E but not by transferase 13; transferase C was not tested. These data were initially obtained by Hayakawa et al. (33) with an assay in which the reaction product with radio- active GSH was isolated by paper chromatography. The exist- ence of the reaction was confirmed by us with the assay described in Table I.

The ability of the transferases to conjugate sulfhydryl ana- logues of GSH was examined. The assay with truns-4-phenyl- 3-buten-2-one was chosen for testing the conjugation of L-cys-

teine, N-acetyl-L-cysteine, and 2-mercaptoethanol because the spectral change in this assay would be expected to be relatively independent of the group adding to the carbon-carbon double bond. Transferases A, B, and C were inactive when these mercaptans replaced equimolar quantities of GSH. With the use of the 1,2-dichloro-4-nitrobenzene assay system, 1 mM homoglutathione was as active as 1 mM GSH with transferases A and C; transferases U and E were not tested.

A summary of selected kinetic constants for transferases A, B, C, and E is presented in Table VII.

DISCUSSION

In view of the physical similarities and the wide and over- lapping substrate specificities of the four homogeneous glutathi- one transferases, the possibility of a relationship among these enzymes must be suspected. Each of the four enzymes is a basic protein of molecular weight 45,000, composed of two sub- units of 25,000 daltons. Transferases A, B, and C were ob- tained in amounts sufficient for amino acid analysis which

allowed calculation of a difference index (34) between them; a value of zero would indicate complete compositional homology, whereas a value of 100 would indicate the total absence of homol- ogy. Comparison of transferase A with transferase C led to a value of 2.2, whereas comparison of transferase A with trans- ferase B and B with C resulted in values of 9.5 and 8.8, respec- tively. Although these numbers represent only statistical evaluations and are not to be taken as definitive in establishing a relationship, the values are much smaller than would be ex- pected by chance and are similar to those found among immuno- globins of the same species.

A definite relationship does exist between transferases A and C, both of which are precipitated by an antibody prepared against transferase A; transferases B and E do not cross-react with this antibody. An antibody active against transferase U and with ligandin (35), the organic anion-binding protein of rat liver, does not react with any of the other three transferases.

In view of the similar physical properties of these enzymes, it is not surprising that earlier investigators, working with rela- tively impure enzyme preparations, ascribed a separate enzyme to each group of substrates, e.g. aryl- or alkyltransferase. Al- though there is an overlap in specificity, the four species studied can be differentiated on the basis of catalytic activity and im- munochemical behavior. Transferases B and E are both active in conjugating iodomethane, a property not shared within ex- perimental limits by transferases A and C; transferase B will not conjugate epoxides and transferase E is only poorly active with I-chloro-2,4-dinitrobenzene. Transferases A and C may be differentiated by the 20-fold greater activity of the latter with trans-4-phenyl-3-buten-2-one.

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We have presented a series of enzymes of similar function but of overlapping specificity that can serve in the detoxication of a large variety of electrophilic compounds, some of which are highly. effective pharmacological agents. An answer to the question of relationship among the glutathione transferases requires isolation of larger quantities of each species in order to evaluate them on the basis of primary structure. Such attempts are in progress.

Acknowledgments-It is with pleasure that we acknowledge

the help and advice of Chaveva Isersky in the immunological

aspects of this study, of Leonard D. Kohn with analytical ultra- centrifugation, and of George Poy in obtaining the amino acid analyses.

REFERENCES

1. BOOTH, J., BOYLAND, E., AND SIMS, P. (1961) Biochem. J. 79, 516324

2. BOYLAND, E., AND CHASSEAUD, L. F. (1969) Advan. Enzymol. 32, 172-219

3. WOOD, J. L. (1970) in Metabolic Conjugation and Metabolic Hydrolysis (FISHMAN, W. H., ed) Vol. 2, pp. 261-299, Aca- demic Press, New York

4. GROVER, P. L., AND SIMS, P. (1964) Biochem. J. 90, 603-606 5. JOHNSON, M. K. (1966) Biochem. J. 98, 44-56 6. CHASSEAUD. L. F. (1973) Biochem. J. 131. 765-769 7. BOYLAND, 6., AND WILLIAMS, K. (1965) kochem. J. 94, 190-

197 8. FJELLSTEDT, T. A., ALLEN, R. H., DUNCAN, B. K., AND JA-

KOBY, W. B. (1973) J. Biol. Chem. 248, 3702-3707 9. PABST, M. J., HABIO, W. H., AND JAKOBY, W. B. (1973) Bio-

them. Biophys. Res. Commun. 62, 1123-1128 10. PABST, M. J., HABIG, W.,H., AND JAKOBY, W. B. (1974) J.

Biol. Chem. 249, 7140-7148 11. JERINA, D. M., DALY, J. W., WITKOP, B., ZALTZMAN-NIREN-

BERG, P., AND UDENFRIEND, S. (1970) Biochemistry 19, 147- 156

12. CARNEGIE, P. R. (1963) Biochem. J. 89, 471-478 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL,

R. J. (1951) J. Biol. Chem. 193, 265-275 14. GILLHAM, B. (1971) Biochem. J. 121, 667-672

15. GOLDSTEIN, J., AND COMBES, B. (1966) J. Lab. Clin. Med. 67, 863-872

16. SNELL, F. D., AND SNELL, C. T. (1949) Calorimetric Methods of Analysis, 3rd Ed, Vol. 2, p. 804, D. Van Nostrand Co., New York

17. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77 18. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-

4412 19. HIRS, C. H. W. (1956) J. Biol. Chem. 219, 611621 20. EDELHOCH, H. (1967) Biochemistry 6, 1948-1954 21. HEBERT, G. A., PELHAM, P. L., AND PITTMAN, B. (1973)

Appl. Microbial. 26, 26-36 22. COHN, E. J., AND EDSALL, J. T. (1965) Proteins, Amino Acids

and Peptides, p. 375, Hafner Publishing Co., Inc., New York

23. WRIaLEY, C. W. (1972) Methods Enzymol. 22, 559-564 24. KABAT, E. A., AND MAYER, M. M. (1961) Ezperimental Im-

munochemistry, 2nd Ed, pp. 85-90, Charles C Thomas, Springfield, Ill.

25. SCHEIDEGGER, J. J. (1955) Znt. Arch. Allergy Appl. Immunol. 7, 103-110

26. JAKOBY, W. B., AND FJELLSTEDT, T. A. (1972) in The Enzymes (BOYER, P. D., ed) 3rd Ed, Vol. 7, pp. 199-122, Academic Press, New York

27. Enzyme Nomenclature, 1972 (1973) Elsevier, Amsterdam 28. BOYLAND, E., AND CHASSEAUD, L. F. (1969) Biochem. J. 116,

958-991 29. SCHOENFIELD, L. J., FOULK, W. T., AND BUTT, H. R. (1964)

J. Clin. Invest. 43, 1409-1418 30. JAVITT, N. B., WHEELER, H. O., BAKER, K. J., RAMOS, 0. L.,

AND BRADLEY, S. E. (1960) J. Clin. Invest. 39, 1570-1577 31. COMBES, B., AND STAKELUM, G. S. (1960) J. CZin. Invest. 39,

1214-1222 32. BEYER, K. H., BAER, J. E., MICHAELSON, J. K., AND Russo,

H. F. (1965) J. Pharmacol. Exp. Ther. 147, l-22 33. HAYAKAWA, T., LEMAHIEU, R. A., AND UDENFRIEND, S. (1964)

Arch. Biochem. Biophys. 162, 223-230 34. METZGER, H., SHAPIRO, M. B., MOSIMANN, J. E., AND VINTON,

J. E. (1968) Nature 219, 1166-1168 35. HABIG, W. H., PABST, U. J., FLEISCHNER, G., GATMAITAN, Z.,

ARIAS, I. M., AND JAKOBY, W. B. (1974) Proc. Nat. Acad. Sci. U. S. A., in press

by guest on Decem

ber 29, 2019http://w

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William H. Habig, Michael J. Pabst and William B. JakobyMERCAPTURIC ACID FORMATION

Glutathione S-Transferases: THE FIRST ENZYMATIC STEP IN

1974, 249:7130-7139.J. Biol. Chem. 

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