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Studies on glutathione-S-arene oxidase transferase—A sensitive assay and partial purification of the enzyme from sheep liver
ARCHIVES
OF BIOCHEMISTRY
Studies Assay
AND
BIOPHYSICS
162, 223-230
(1971)
on Glutathione-S-Arene
Oxidase
and
of the
Partial
TL1RO HAYAKAWA,2 l
of Molecular
Purification RONALD Biology
Transferase’-A Enzyme
A. LERIAHIEU,
AND
SIDSEY
and Chemical Research Department, Nutley, iVew Jersey OYllO
Received
September
from
Sensitive Sheep
Liver
UDENFRIENDS
Hofltrlan-La
IZoche. Inc.,
24, 1973
A sensitive assay has been devised for glutathione-S-arene oxidase transferase using as substrates naphthalene-1,2-oxide or styrene oxide along with [%]glutathione. Activity of the order of 2-3 nmoles of conjugate formed during a 5-min incubaiion can be detected. This yields about 2000 cpm above a blank of about 1500 cpm. Transferase activity was found mainly in liver and kidney but was also present in most other tissues of rats. Glutathione-S-arene oxide transferase has been purified 70- to 80-fold from sheep liver 100,000 g supernatants using the conventional procedures. After electrofocusing, enzyme activity separated into two major peaks and 1.~0 or three minor peaks, ranging in isoelectric point from pH 6.5 to 7.5. iictivities assayed with naphthalene-1,2-oxide or styrene oxide as substrates were found to almost parallel each other in all the peaks. The sheep liver transferase required neither metal ions nor cofactors such as FAI), pyridoxal-phosphate and thiamine pyrophosphate. The molecular weight of thr transferase has been estimated to be about 40,000. K, values for glutathione, naphthalene-1,2-oxide, and styrene oxide are 1.6, 0.11, and 0.13 mM, respectively. K, values for glutathione decreased with increasing pH, whereas the K, values for naphthalene-1,2-oxide were independent of pH in the range of 6.5-8.
(3, 5, S, 9). Arcane oxides rearrange nonenzymatically to phenols (3, 5) and ma) react with macromolecular tissue (JOIIstituents like nucleic acids and some specific proteins (10-12). Recently a glutathione& epoxide transferase has been purified from 100,OOOgsupernatant of rat liver. This cnzyme was found to be active only mit,h alkyl epoxides suggesting the presenw of another enzyme which catalyzes rchactions with arene oxides (13). In order to look for glutathione-S-arene oxide transferase activity, a sensitive assay was devised with either naphthalme-1,2-oxide or styrcwe oxide and [35S]glutathione as substrates (Fig. 1). Wit,h the newly devised sensitivr assay, glutathione-S-arene oxide transfcrasc activit’y has been purified 70- to SO-fold from sheep liver 100,OOOgsupernatants. Dtbi ail*
Alkyl cpoxidcs and arene oxides have been idmt,ified as obligatory intermediates in hydroxylation of certain foreign compounds catalyzed by hepatic P-4.50.dependent monooxygenases (2, 3). The following two pathways have been recognized as key metabolic steps in the detoxification of these intermediary metabolites: (a) hydration of the oxide to the corresponding diol by hepatic epoxide hydrase(s) (3-7) and (b) conjugation with glutathione 1 A preliminary report of this work has been presented (I ). 2 Present address: Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464, Japan. 3 To whom reprint requests should be addressed. 223
224
HAYAKAWA,
FIG. 1. Enzymatic
LEMAHIEU,
formation of glutathione
of the new assay and the partial purification of glutathlone-S-arene oxide transferase from sheep liver arc presented in this paper. MATERIALS
AND METHODS
[3%]Glutathione (500 pCi/2.45 mg), radiochemical purity >99% was purchased from Schwarz/ Mann and nonradioactive glutathione from Sigma and Aldrich Chemicals. Tetrapotassium pyrophosphate and sodium acid pyrophosphate were kindly supplied by Stauffer Chemical Co. DEAEcellulose (DE-52) and CM-cellulose (CM-52) were purchased from Whatman. Hydroxylapatite (Bio-GEL HT) from Bio Rad Laboratories. Sephadex G-75 from Pharmacia Fine Chemicals. Naphthalene-1,2-oxide was synthesized from 4-bromo-1,2-epoxy-1,2,3,4-tetrahydronaphthalene by the method reported by Vogel and Klarner (14). Crystallization by cooling at -80°C yielded the pure oxide which gave an nmr spectrum identical with that reported by Vogel and Kliirner. Tic on silica gel using benzene(3:3:chloroform-ethyl acetate-triethylamine 3:0.5) as solvent showed a single spot (Rf 0.56). No cu-naphthol was detected. 7-Tritiated styrene oxide was kindly supplied by Dr. John W. Daly and 2-tritiated naphthalene-1,2-oxide was kindly supplied by Dr. Donald M. Jerina (N.I.H., Bethesda, MD). Liver supernatants were prepared from 25% (w/v) homogenates in 0.1 M potassium phosphate buffer, pH 7.4 by centrifuging at 23,500 9 for 25 min and subsequently at 100,OOOgfor 2 hr. Incubation mixtures contained 0.5 pmole of [%]glutathione (4-5 X lo6 cpm), 100 pmoles of pyrophosphate buffer pH 8, 0.6 Hmole of naphthalene-1,2-oxide or styrene oxide in 2 ~1 of ethanol, and enzyme in a total volume of 1 ml. The reaction was started by the addition of naph-
AND UDENFRIEND
conjugate with naphthalene-1,2-oxide. thalene-1,2-oxide or styrene oxide. After incubation at room temperature (22°C) for 5 min, the reaction was terminated by the addition of 50 ~1 of 4 N acetic acid. The conjugates produced during the incubation were adsorbed on 20 mg of activated charcoal which was collected by centrifugation. After washing the charcoal with 6 ml of water, the conjugates were eluted twice with 1 ml of methanol-benzene-aqueous ammonia (87:10:3) as previously described (3). The combined eluates were evaporated under nitrogen, the residues were dissolved in 40 ~1 of 50% ethanol and applied to Whatman No. 3 MM paper. Chromatograms were developed in a descending manner with 1 butanol-acetic acid-water (12:3:5). The bands on each chromatogram corresponding to the conjugate (R, = 0.3 for both conjugates), were made visible on the paper by exposure to ultraviolet light (254 nm) or by spraying with ninhydrin (Fig. 2). The latter procedure was required when the conjugate contained no aromatic substituents, as with styrene oxide. The appropriate bands were cut out and transferred to a vial containing 1 ml of 5Oyo ethanol. After standing for about 15 min, to permit extraction of the conjugate, 10 ml of Bray’s solution was added and radioactivity was measured in a scintillation counter. Boiled enzyme preparations (5 min, 1OO’C) or incubations carried out in the absence of enzyme served as controls. In some instances chromatograms were scanned for radioactivity in a Packard Radiochromatogram Scanner Model
385. For measuring transferase activity with 2tritiated naphthalene-1,2-oxide or ‘I-tritiated styrene oxide, with nonradioactive glutathione as substrate, incubation mixtures contained the indicated amounts (see figure legends) of [2-3H]naphthalene-1,2-oxide (3 X 105 cpm/0.6 pmole)
FIG. 2. Chromatography of glutathione conjugate with naphthalene-1,2-oxide (A) reduced form of glutathione (B). Chromatograms were first examined under uv light the absorbing bands traced in pencil. They were then treated with ninhydrin to yield characteristic color. A minor spot (R, = 0.05) close to the origin was identified as the dized form of glutathione.
and and the oxi-
(;SH-S-ARENE
OXIDE
or [‘iJH]styrene oxide (4-5 X lo5 cpm/l.2 pmoles); 100 ~moles of pyrophosphate buffer, pH 8, with 5 pmoles of gllltathione or 100 rmoles of pyrophosphate buffer, p1I 6.5, with 30 rmoles of gl\ltathione and enzyme in 1 ml of total volume. To prepare sheep liver 100,OOOg supernatants for rllzynlr purification, fresh sheep liver from a slaughterhouse was homogenized with 3 vol of ice,-cold 0.1 M potassium phosphate buffer, pH 7.4, ill a Waring Blendor for 45 sec. The homogenatc’ was first rentrifuged in a Sorvall (; 3 rotor for 25 min at 8000 rpm (10,825g). The resultant supernat :tnf, was centriftlged again in a Beckman 4”.1 rotor for 2 hr at 40,000 rpm (100,OOOg). The supernatant was collect,ed and stored in a deepfreeze for frlrther purification. Protein was det,ermined I)>- the J,owry method (15). However, for colu~nn elnatcs, the flllorescamine method was nsed IO conserve material and time (16). I*;lrct roforllsing st lldies were carried out in an LKH 110.ml elcctrofocllsing col\lmn. A 1’;; ampholitlr Folllt.ion of pH range 5-8 was used with a 0 X”, (v v) continllous sllcrose gradient supplenrrntrd with 30’( glycerol. The protein solution, 11 rug protein from Sephadex (i-75 column fractions, (see below) was added to the light solution to establish t.hr gradient. The voltage was increased from an initial value of 800 V to 1000 V over the collrse of 2 days, and focusing was continrleti for an additional 4 days. The clirrent fell front 2 nlri to 0.5 11~4 over this period. Ethylenedianrillc was llsed as the cathode solution (top) and phosphoric acid as the anode solution (bottom ). The contents of the colunn were pumped ont wit11 a11 LKB l’erpex Peristaltic pump and 0%1111 fractions were collected. The plI of the Ilrldilrttcd fractions was determined at 4°C with a 13cv*kl11:trr b2odel i(i I)igital p1-I meter. .\c~ryl:unide gel elertrofocusing studies were carricstl out rising 1’ ( ampholine in the pH range of 5 8 with T.S’, acrylamide gel. A 25.~1 (860 rg prolc.in ) sample of sheep liver 100,OOOg supernat,:lllt was nlixed with acrylamide-bisacrylamide solrrt ion , and photopolymerized in the presence of riboflavin (17). Cllrrent was applied at 75 V for L’O III. at 4°C:. I~;t,hylenediamine, 1“; (v/v), was IISWI as the cathode solution (top) and 1.25ci phosphoric acid as thra anode solut,ion (bottom). After tllis thr gel was frozen and cut into l-mm slices. I,::rch slice was transferred to a small test t rrhe containing 0.2 ml of water and extracted for IGI hr at 0°C. The pI1 was determined at 4°C using a thill combination electrode. Each tube with the gel slice still present was subjected to glutathioneS-arckricx oxide transferase assay lising 3 /Imoles of t~a~~httl:dt=rre~l ,%-oxide or styrene oxide instead assay, and inof O.(i ~molr as in the standard cllt):lt~~~tl for 20 mill at roon1 temperature (22%).
.)‘),,.I
TIZANSFE;l’,ASP; These were not the best kinetic were used to increase sensitivity parative stlldy. 1: I’SULTS
ANI)
conditions for this
but, corn-
L)ISCUSSION
Two mcathods for mctasuring glutathionc transfwaw activity \\-(w considcwd (13 1; a spt~ctrophotomctric: assay bawd on the increw of absorbance at 330 1~11 upon glutathiow conjugat’ion with 1 ,2-~poxy-:<(in-rlitroph(~nox~~ propaw, :trd :L caolorimrtric assay bawd on the mcasuwmt~nt of the dccrwscb in ~oticclnt,ratioIl of th(b sulfhydryl groups of glutathione as dt>twmincd by thr waction with 5 ,.5’-dithiobis (2.nitrobcnzoatr). Both assays arch rapid and simplr but inscwGtivc1 and thcl former is limitc~tl to a single alkyl clxpoxidc substratcb. A4now radioassay was dcvcIopcd bawd on the formation of radioactive glutathiotw conjugate from naphthalrtwl ,L’-oxidt~ and [“%]glutathioncl (Fig. 1). liecowry of thcl thcl proccdurcb \\-as conj ugat (L through chcckrd by using a standard [“%]glut:~naphthalsw-1 ,2thiow cotijugatch with oxide Lvhich had hcen sgnthwixtd mzy,1 certain amount (front Z-11 matically. nmolcs) of th(x [Y3]gluthathiow conjugate, synthcsixcd as mentioned above, KM added to a reaction mixture which was twutcd as :I st,andard assa\-. Tlw radioactivity \vhicah \\-as rwovcwd ~-as cwmparc~d \vi th the> original radioactivity. Rwowricw avctragcd 52.5 * 0.0 “i. :lssx~~ valurs prrwntc~d ill this papor wrc not corrcrtod for rcwwcyy. The ticn- assay is quite: swsitiw (3w1 though th(a rcwwwy of product is SOI~W\\-hat low. An important advantagc~ is that t#ho assay is applicabh~ to most a1lq.I qoxides and awnc oxides. ‘l’hc~ aesw\- has t h(> advantage of giving clualitutiw c~onfirnlntion of thtl formation of conjugate on p:tp~ chromatograms prior to c>lution and radioactive mcasur(wwt (E‘ig. ‘2). Thcx appropriatc spot,s on chromat~ograms wro identific>d as the glutathiotw conjugatcl \vith r~phthal(w-1 ,L’-osidcl by thrlir positiw wwtion wit’h l<~Cr~O~-AgSO:~ wagc~nt~(IS), by tAicGr absorption of’ short-\~LLvc~lctlgtl-I uv light and by thAr wac+ion nith ninhydrin. Th(l variat,ioti :+ctivit\. of transfrraw with pH is shon-11 in Fig. 3. Thr~ cwzymat,ic wact,ion VW maximal at about S.(i. Ho\v-
226
HAYAKAWA,
Tl2.-
?
I
’
E
I
I
LEMAHIEU, I
I _
_ IO-
AND UDENFRIEND
yielded about 2000 cpm above a blank of about 1500 cpm. Even at 22°C conjugate formation was not linear for more than 5 min, as shown in Fig. 5. In order to maintain initial rates a 5-min incubation period was used in the assay. The effects of glutathione concentration on both the enzymatic and the nonenzymatic formation of conjugate between glutathione and naphthalenc-1,2-oxide are shown in Fig. 6. Conjugate formation in the presence of enzyme exhibited the typical saturation kinetics of an enzyme-catalyzed reaction. The nonenzymatic formation of
PH
curves of gluFIG. 3. (A) The pH activity tathione conjugate formation with naphthaleneenzymatic (47 pg of a CM1,2-oxide. -•---: cellulose column fraction of the sheep liver transferase) and -C--: nonenzymatic. In this and subsequent figures values from the enzymatic and nonenzymatic reactions were all corrected for the blank obtained in the absence of naphthalene1,2-oxide. The enzymatic formation of conjugate was further corrected to obtain the net value by subtracting the value from the nonenzymatic formation of conjugate. Details of the assay conditions are described under Methods. TIME 20-
0
(min)
5. Time course of the formation of glutathione conjugate with naphthalene-1,2-oxide at room temperature (22°C). --•---: enzymatic (107 kg of a DEAE-cellulose column of sheep liver transferase) and -C---: nonenzymatic.
IO
20
30
pg PROTEIN FIG. 4. Transferase activity as a function of protein concentration. A Sephadex G-75 column fraction of sheep liver transferase was used with 402,800 epm of [%]glutathione.
ever, the nonenzymatic reaction increased markedly above pH 8. Incubations were carried out at pH 8 to minimize the nonenzymatic reaction. Conjugate formation as a function of enzyme concentration is shown in Fig. 4. Activity of the order of 2-3 nmoles of conjugate formed during 5 min of incubation
0
2 GSH
I I I I 4 6 CONCENTRATION
I 8
I IO (mM)
6. Effects of glutathione concentration on glutathione conjugate formation with naph-•-: enzymatic (22.8 thalene-1,2-oxide. rg of Sephadex G-75 column fraction of sheep liver transferase) and -C--: nonenzymatic. FIG.
GSH-S-ARENE
OXIDE
conjugate required much higher concentrations of glutathione and exhibited secondorder reaction kinetics, bypical of a pure chemical reaction. Enzymatic activit’y was shown to be destroyed by tryptic digestion and the tryptic effect could be abolished by the addition of soybean trypsin inhibitor. Since significant conjugation between glutathione and arene oxides or alkyl epoxides can proceed nonenzymatically, it was necessary to SW whether nonspecific protcins could catalyze the react’ion. To invcst’igate this, 0.5 mg each of bovine serum albumin, aldolase, L-glutamic acid dehydrogenase, a-globulin, and fibrinogen wre examined for t’ransferase act’ivity. Sorw of these proteins exhibited activity above the controls. The transferase activity was found mainly in liwr and kidney of adult rats, but was also present in many other organs, including skeletal muscle and skin (Table I). Transfcrase activities in liver 100,OOOg suprrnatants from various other species TABLE
I
DISTRIBUTION OF GLUTATHIONE-S-ARENE OXIDE TRANSFERASE ACTIVITY IN VARIOUS RAT TISSUES Organ” Activity (nmoles conjugate formed/g wet wt/min) Cerebrum Cerebellum Brain stem Lung Heart Liver Kidney Spleen Skeletal muscle Skin Small intestine Stomach -
31 34 36 37 19 387 191 30 16 9b 30 30
a Tissues of two adult male Sprague-Dawley rats weighing 500 g each were combined for the experiment. Naphthalene-1 , Z-oxide was used as substrate. b A 0.4.ml aliquot of l~,~Og supernatant from 25% skin homogenate showed 3200 cpm above a blank of about 2500 cpm. All other tissues gave proportionally higher radioactivity.
WY
TRANSFERAHI~: TABLE
II
GLUTATHIONE-S-ARENE OXIDE TR.INSFE;R.\~IC :'uJTIVITIESINLIVER 100,000g SUPERN.ITANTS FROM V.~MOUS SPECIES" Species
Activities (nmoles conjugate formed/g wet wt {min) _~ .-__ With With styrene ouidt naphthalene1,2-oxide
Sheep Horse (frozen) Cattle Hog Human (male, autopsy) Monkey (frozen) Rabbit (frozen) Guinea pig Rat (male) Rat (female) Mouse (male) Mouse (female)
96 152 438 228 187 665 327
f f f f
43 26 94 16
1,562 969 2 2-11 11992
It f Zt *
175 155 217 216
a Details of the preparation of liver IW,OoOg supernatante are described under Methods. b The values represent the mean f 81) oi Four sheep, six male and female Sprague-Dawley rats; five male and female Carworth Farm mice. In all other cases the values represent assays ~)blainrd on tissues from one animal.
were also examined (Table II). An intwcbst ing finding was the significant diffcrcwc bctwcen male and female mice. 1lalc mice showed twice as much activitv as fcwal(l mice with naphthalene-1 , Zoxidc as hub strate; however, with styrene oxidcl as wb strate, there was little diffcrencc br:t\vccn the sexes. Male rats exhibited a somewhat higher activity than female rats lvith both napht’halene-1 , Z-oxide and styrcwb oxide as substrates. The nat’ure of tht>se diffcrorwes is under investigation. All the procedures for t’hc purification4 of glutathione-S-arenc oxide tr:lnsfrrasr act,ivity UYW carried out bet wwl 0” :1nt1 4 Thrl purificat,ion procedure was indt:pt~lttlently developed in this laboratory. W’hilf~ this manuscript was being prepared, a pro~durt~ t’or the purification of rat liver glutathiotle-~‘-alkyl epoxide transferase was made known to us (13) The two procedures were developrd indf~pt~ncl ent,lq- and the similarities are fort,uitorlF
228
HAYAKAWA,
LEMAHIEU,
4°C. Sheep liver supernatant, 500 ml, was adjusted to pH 5.4 with 5 % acetic acid, and the precipitate was removed by centrifugation at 9000 rpm (13,000g) for 45 min in a Sorvall G S rotor. The resultant supernatant was adjusted to pH 7 with 1 M potassium phosphate buffer, pH 7.4, and subjected to ammonium sulfate fractionation. Enzyme activity was precipitated between 20 and 65%, (w/v) of ammonium sulfate. The precipitate was collected by centrifugation and dissolved in a minimal amount of 1 mM potassium phosphate buffer, pH 7.4, and dialyzed for 24 hr against two 4-lit’er changes of the same buffer. The dialyzed fraction (148 ml) was further purified on a DEAE-cellulose column (2.ti X 34 cm) equilibrated with 1 mM potassium phosphate buffer, pH 7.4. The enzyme activity appeared in the void volume on washing with 5 mM phosphate buffer, pH 7.4. The active fractions were combined (140 ml) and dialyzed overnight against 4 liters of Yj mM phosphate buffer, pH 6.5. The dialyzed preparation was next applied to a CR/I-cellulose column (2.5 X 20 cm) which had previously been equilibrated with 5 mM phosphate buffer, pH 6.5. Again the active protein appeared in the void volume on washing with the same buffer. The active fractions (137 ml) were applied to a hydroxylapatite column (2.5 X 9.5 cm) equilTABLE PURIFICATION
OF GLUTATHIONE-S-ARENE
Liver supernatant (100,OOOg) pH 5.4 supernatant Ammonium sulfate fractionation DEAE-cellulose column CM-cellulose column Hydroxylapatite column Sephadex G-75 column Electrofocusingb Peak I Peak II
(20-65yc)
AND
UDENFRIEND
ibrated with 5 mM phosphate buffer, pH 6.5. After washing with 10 mM phosphate buffer, pH 6.5 (ca. 325 ml), the column was subjected to a linear gradient from 10 mM to 100 mM phosphate buffer, pH 6.5 (300 ml in each reservoir). The active protein appeared in the fractions which were 50-60 mM in phosphate concentration. Active fractions from the hydroxylapatite column were pooled and concentrated by ammonium sulfate fractionation ((r65 %), and the precipitate was dissolved in about 7 ml of 1 mM phosphate buffer, pH 7.4. The solution was applied to a Sephadex G-75 column (2.5 X 95 cm) equilibrated with 5 mM phosphate buffer, pH 6.5, containing 0.1 N NaCl, and the column was then washed with the same buffer. Peak fractions were collected and stored in a freezer. The results of the purification of the transferase from sheep liver are summarized in Table III. As a final step, 11 mg protein from the peak fractions of the Sephadex G-75 step were subjected to electrofocusing on a column containing 1% ampholine of pH range 5-8. The results of the electrofocusing are shown in Fig. 7. Enzyme activity separated into several forms, two major peaks and two to three minor peaks, ranging in isoelectric point from pH 6.5 t’o 7.5. Furthermore, activities assayed with both naphthalene-l,2-oxide and styrene oxide as III
OXIDE TR~INSFERME
ACTIVITY
Protein bd
Sp acta
17,400 9,943 6,290 1,848 771 261 101
4.2 5.8 7.0 24.4 45.0 102.0 192.9 292.6 332.0
FROM SHEEP LIVEI~ Yield (%I 100 80 61 tY2 48 37 27
Purification factor 1 1.4 1.7 5.8 11 24 46 70 79
Q With the standard assay using naphthalene-1,2-oxide as substrate and expressed as nmoles of conjugate formed/mg/min. h The activities of the two major peak fractions are shown; Peaks I and II correspond to I and II in Fig. IV.
GSH-S-ARENE
OSII>E
229
TRANSFP:RiZSl~:
4 80C
-460C a 5 0 '-
‘,
0i
3
i 20
40
60
Fractions
80 (0.8
100
120
ml/tube)
FIG. 7. Isoelectric focusing of glutathione-S-arene oxide transferase. Theenzyme protein (11 mg) from the Sephadex G-75 column preparation was applied to the column and r,ln as described in Methods. Peaks I and II correspond to those shown in Table III.
suhstratcs wert~ found to almost parallel ctach other in all the peaks. Further invcstigation of these multiple forms were carried out’ on acrylamide gel clectrofocusing n-it,h crude extracts of sheep liver. Similar multiple forms, two major peaks and two to thrw minor peaks, w-err found in crude c>xtracts. Disc gel electrophoresis of the peak II fraction from the clectrofocusing column, by the method of Ornstcin (19) and Davis (20), rcwaled two major protein bands (about :$O and 50% of total protein by Coomassicl blue staining) which ran close together. It’ was shown that the protein bands were catalytically active, but it was not possihlc to sholz- which of the two prot&l bands on the gcbl was active. Furhher purification is in progrcw to obtain a homogeneous preparation. Thfl molrcular wcxight of th(t partially tw-ific>d transf(lraw (after the hydroxylapatite column stctp) I\-as determined by using a calibrated Scphadcx G-75 column and wt,imatcld (‘1) as 40,000. This valw is
Substrates
(ilutathione Naphthalene-1,2oxide Styrene oxide
K, values (nm) pH 6.5
pH i
pH X
10 0.08
5
I .ti 0.1 I 0. 1:3
R K, values for glut,athione were determlned with a naphthalene-1,2-oxide concentration of 0.6 mM. K,, values for naphthalene-1,2-oxide and styrene oxide were determined using 2-tritiated or 7-tritiated styrtrne naphthalene-1,2-oxide oxide, and nonradioactive glutathionr as slit)strates. The glutathione concentjration was 5 111~. Details of the assay conditions are desc&ribed urrde~ Methods.
230
HAYAKAWA,
LEMAHIEU,
Enzyme preparations after Sephadex gel filtration were fairly stable on storage at -90°C. They gradually lost activity over a period of a few months. The effect of divalent cations on transferase activity was examined and it was found that 2 mM CaC12, MgC&, and ZnCL, neither activated nor inhibited. However, 2 mM CuSO4 caused 73% inhibition. Transferase activity was not inhibited by 2 mM EDTA and was not activated by 0.1 mlvI FAD, pyridoxal phosphate, or thiamine pyrophosphate. K, values for both glutathione and naphthalene-1,2-oxide, determined at different pH values, are summarized in Table IV. K, values for glutathione decreased with increasing pH, whereas the K, values for naphthalene-1,2-oxide were the same at two different pH values, 6.5 and 8. Similar results have been reported in the case of the rat liver enzyme (13). The K, value for glutathione at pH 8 was calculated as 1.6 mM; however, for the standard assay system 0.5 mM glutathione was used to increase the sensitivity of the assay and also t’o minimize the nonenzymatic formation of conjugate. Sheep liver transferase is highly specific with respect to glutathione; however, the enzyme accepts many different oxides as substrates. Details of substrate specificity and structure-activity relationships of a large number of arene oxides and alkyl epoxides will be discussed in a subsequent communication (22). Recently, one of the purest preparations of rat liver glutathione-S-alkyl epoxide transferase obtained by Fjellstedt et al. (13) was supplied to us by Dr. W. B. Jakoby and was found to contain comparable amounts of transferase activity, using the new sensitivc assay system with naphthalene-1 ,2oxide as substrate. It appears now that the failure to observe arene oxide activity in their preparations (13) was due to a lack of sensitivity and reproducibility of the method used to detect arene oxide-glutathione conjugates.5 5 Personal communication Jakoby, National Institutes MD.
from Dr. W. B. of Health, Bethesda,
AND
UDENFRIEND ACKNOWLEDGMENTS
We acknowledge the help and advice of Dr. Kenneth Gibson with the work involving electrofocusing. REFERENCES 1. HAYAKAWA, T. (1973) Fed. Proc. 32, 666. 2. LEIBMAN, K. C., AND ORTIZ, E. (1970) J. Pharmacol. Exp. Ther. 173, 242. 3. JERINA, D. M., DALY, J. W., WITKOP, B., ZALTZMAN-NIRENBERG, P., AND UDENFRIEND, S. (1970) Biochemistry 9, 147. 4. MAYNERT, E. W., FOREMAN, R. L., AND WATABE, T. (1970) J. Biol. Chem. 246, 5234. 5. JERINA, D. M., DALY, J. W., WITKOP, B., ZALTZMAN-NIRENBERG, P., AND UDENFRIEND, S. (1968) Arch. Biochem. Biophys. 128, 176. 6. JERINA, D. M., ZIFFER, M., AND DALY, J. W. (1970) J. Amer. Chem. Sot. 92, 1056. 7. OESCH, F., JERINA, D. M., AND DALY, J. W. (1971) Arch. Biochem. Biophys. 144, 253. 8. BOYLAND, E., AND WILLIAMS, K. (1965) Biothem. J. 94, 190. 9. GROVER, P. L., HEWER, A., AND SIMS, P. (1973) Fed. Eur. Biochem. Sot. Lett. 34, 63. 10. GROVER, P. L., AND SIMS, P. (1970) Biochem. Pharmacol. 19, 2251. 11. GROVER, P. L., FORRESTER, J. A., AND SIMS, P. (1970) Biochem. Pharmacol. 20, 1297. 12. KUROKI, T., AND HEIDELBERGER, C. (1972) Biochemistry 11, 2116. 13. FJELLSTEDT, T. A., ALLEN, R. H., DUNCAN, B. K., AND JAKOBY, W. B. (1973) J. Biol. Chem. 248, 3702. 14. VOGEL, E., AND KLXRNER, F. G. (1968) Angezu. Chem. Int. Ed. Engl. 7, 374. 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND R.aNDALL, R. J. (1951) J. Biol. Chem. 193, 265. 16. BOHLEN, P., STEIN, S., DAIRMAN, W., AND UDENFRIEND, S. (1973) Arch. Biochem. Biophys. 166, 213. 36, 17. WRIGLEY, C. W. (1968) J. Chromatogr. 362. 18. KNIGHT, R. H., AND YOUNG, L. (1958) Biothem. J. 70, 111. 19. ORNSTEIN, L. (1964) Ann. N. Y. Acad. Sci. 121, 321. 20. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404. 21. DETERMANN, H. (1968) Gel Chromatography, 2nd ed., p. 110, Springer-Verlag, New York. 22. HAYAKAWA, T., UDENFRIEND, S., YAGI, H., AND JERINA, D. M., In preparation.
OF BIOCHEMISTRY
Studies Assay
AND
BIOPHYSICS
162, 223-230
(1971)
on Glutathione-S-Arene
Oxidase
and
of the
Partial
TL1RO HAYAKAWA,2 l
of Molecular
Purification RONALD Biology
Transferase’-A Enzyme
A. LERIAHIEU,
AND
SIDSEY
and Chemical Research Department, Nutley, iVew Jersey OYllO
Received
September
from
Sensitive Sheep
Liver
UDENFRIENDS
Hofltrlan-La
IZoche. Inc.,
24, 1973
A sensitive assay has been devised for glutathione-S-arene oxidase transferase using as substrates naphthalene-1,2-oxide or styrene oxide along with [%]glutathione. Activity of the order of 2-3 nmoles of conjugate formed during a 5-min incubaiion can be detected. This yields about 2000 cpm above a blank of about 1500 cpm. Transferase activity was found mainly in liver and kidney but was also present in most other tissues of rats. Glutathione-S-arene oxide transferase has been purified 70- to 80-fold from sheep liver 100,000 g supernatants using the conventional procedures. After electrofocusing, enzyme activity separated into two major peaks and 1.~0 or three minor peaks, ranging in isoelectric point from pH 6.5 to 7.5. iictivities assayed with naphthalene-1,2-oxide or styrene oxide as substrates were found to almost parallel each other in all the peaks. The sheep liver transferase required neither metal ions nor cofactors such as FAI), pyridoxal-phosphate and thiamine pyrophosphate. The molecular weight of thr transferase has been estimated to be about 40,000. K, values for glutathione, naphthalene-1,2-oxide, and styrene oxide are 1.6, 0.11, and 0.13 mM, respectively. K, values for glutathione decreased with increasing pH, whereas the K, values for naphthalene-1,2-oxide were independent of pH in the range of 6.5-8.
(3, 5, S, 9). Arcane oxides rearrange nonenzymatically to phenols (3, 5) and ma) react with macromolecular tissue (JOIIstituents like nucleic acids and some specific proteins (10-12). Recently a glutathione& epoxide transferase has been purified from 100,OOOgsupernatant of rat liver. This cnzyme was found to be active only mit,h alkyl epoxides suggesting the presenw of another enzyme which catalyzes rchactions with arene oxides (13). In order to look for glutathione-S-arene oxide transferase activity, a sensitive assay was devised with either naphthalme-1,2-oxide or styrcwe oxide and [35S]glutathione as substrates (Fig. 1). Wit,h the newly devised sensitivr assay, glutathione-S-arene oxide transfcrasc activit’y has been purified 70- to SO-fold from sheep liver 100,OOOgsupernatants. Dtbi ail*
Alkyl cpoxidcs and arene oxides have been idmt,ified as obligatory intermediates in hydroxylation of certain foreign compounds catalyzed by hepatic P-4.50.dependent monooxygenases (2, 3). The following two pathways have been recognized as key metabolic steps in the detoxification of these intermediary metabolites: (a) hydration of the oxide to the corresponding diol by hepatic epoxide hydrase(s) (3-7) and (b) conjugation with glutathione 1 A preliminary report of this work has been presented (I ). 2 Present address: Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464, Japan. 3 To whom reprint requests should be addressed. 223
224
HAYAKAWA,
FIG. 1. Enzymatic
LEMAHIEU,
formation of glutathione
of the new assay and the partial purification of glutathlone-S-arene oxide transferase from sheep liver arc presented in this paper. MATERIALS
AND METHODS
[3%]Glutathione (500 pCi/2.45 mg), radiochemical purity >99% was purchased from Schwarz/ Mann and nonradioactive glutathione from Sigma and Aldrich Chemicals. Tetrapotassium pyrophosphate and sodium acid pyrophosphate were kindly supplied by Stauffer Chemical Co. DEAEcellulose (DE-52) and CM-cellulose (CM-52) were purchased from Whatman. Hydroxylapatite (Bio-GEL HT) from Bio Rad Laboratories. Sephadex G-75 from Pharmacia Fine Chemicals. Naphthalene-1,2-oxide was synthesized from 4-bromo-1,2-epoxy-1,2,3,4-tetrahydronaphthalene by the method reported by Vogel and Klarner (14). Crystallization by cooling at -80°C yielded the pure oxide which gave an nmr spectrum identical with that reported by Vogel and Kliirner. Tic on silica gel using benzene(3:3:chloroform-ethyl acetate-triethylamine 3:0.5) as solvent showed a single spot (Rf 0.56). No cu-naphthol was detected. 7-Tritiated styrene oxide was kindly supplied by Dr. John W. Daly and 2-tritiated naphthalene-1,2-oxide was kindly supplied by Dr. Donald M. Jerina (N.I.H., Bethesda, MD). Liver supernatants were prepared from 25% (w/v) homogenates in 0.1 M potassium phosphate buffer, pH 7.4 by centrifuging at 23,500 9 for 25 min and subsequently at 100,OOOgfor 2 hr. Incubation mixtures contained 0.5 pmole of [%]glutathione (4-5 X lo6 cpm), 100 pmoles of pyrophosphate buffer pH 8, 0.6 Hmole of naphthalene-1,2-oxide or styrene oxide in 2 ~1 of ethanol, and enzyme in a total volume of 1 ml. The reaction was started by the addition of naph-
AND UDENFRIEND
conjugate with naphthalene-1,2-oxide. thalene-1,2-oxide or styrene oxide. After incubation at room temperature (22°C) for 5 min, the reaction was terminated by the addition of 50 ~1 of 4 N acetic acid. The conjugates produced during the incubation were adsorbed on 20 mg of activated charcoal which was collected by centrifugation. After washing the charcoal with 6 ml of water, the conjugates were eluted twice with 1 ml of methanol-benzene-aqueous ammonia (87:10:3) as previously described (3). The combined eluates were evaporated under nitrogen, the residues were dissolved in 40 ~1 of 50% ethanol and applied to Whatman No. 3 MM paper. Chromatograms were developed in a descending manner with 1 butanol-acetic acid-water (12:3:5). The bands on each chromatogram corresponding to the conjugate (R, = 0.3 for both conjugates), were made visible on the paper by exposure to ultraviolet light (254 nm) or by spraying with ninhydrin (Fig. 2). The latter procedure was required when the conjugate contained no aromatic substituents, as with styrene oxide. The appropriate bands were cut out and transferred to a vial containing 1 ml of 5Oyo ethanol. After standing for about 15 min, to permit extraction of the conjugate, 10 ml of Bray’s solution was added and radioactivity was measured in a scintillation counter. Boiled enzyme preparations (5 min, 1OO’C) or incubations carried out in the absence of enzyme served as controls. In some instances chromatograms were scanned for radioactivity in a Packard Radiochromatogram Scanner Model
385. For measuring transferase activity with 2tritiated naphthalene-1,2-oxide or ‘I-tritiated styrene oxide, with nonradioactive glutathione as substrate, incubation mixtures contained the indicated amounts (see figure legends) of [2-3H]naphthalene-1,2-oxide (3 X 105 cpm/0.6 pmole)
FIG. 2. Chromatography of glutathione conjugate with naphthalene-1,2-oxide (A) reduced form of glutathione (B). Chromatograms were first examined under uv light the absorbing bands traced in pencil. They were then treated with ninhydrin to yield characteristic color. A minor spot (R, = 0.05) close to the origin was identified as the dized form of glutathione.
and and the oxi-
(;SH-S-ARENE
OXIDE
or [‘iJH]styrene oxide (4-5 X lo5 cpm/l.2 pmoles); 100 ~moles of pyrophosphate buffer, pH 8, with 5 pmoles of gllltathione or 100 rmoles of pyrophosphate buffer, p1I 6.5, with 30 rmoles of gl\ltathione and enzyme in 1 ml of total volume. To prepare sheep liver 100,OOOg supernatants for rllzynlr purification, fresh sheep liver from a slaughterhouse was homogenized with 3 vol of ice,-cold 0.1 M potassium phosphate buffer, pH 7.4, ill a Waring Blendor for 45 sec. The homogenatc’ was first rentrifuged in a Sorvall (; 3 rotor for 25 min at 8000 rpm (10,825g). The resultant supernat :tnf, was centriftlged again in a Beckman 4”.1 rotor for 2 hr at 40,000 rpm (100,OOOg). The supernatant was collect,ed and stored in a deepfreeze for frlrther purification. Protein was det,ermined I)>- the J,owry method (15). However, for colu~nn elnatcs, the flllorescamine method was nsed IO conserve material and time (16). I*;lrct roforllsing st lldies were carried out in an LKH 110.ml elcctrofocllsing col\lmn. A 1’;; ampholitlr Folllt.ion of pH range 5-8 was used with a 0 X”, (v v) continllous sllcrose gradient supplenrrntrd with 30’( glycerol. The protein solution, 11 rug protein from Sephadex (i-75 column fractions, (see below) was added to the light solution to establish t.hr gradient. The voltage was increased from an initial value of 800 V to 1000 V over the collrse of 2 days, and focusing was continrleti for an additional 4 days. The clirrent fell front 2 nlri to 0.5 11~4 over this period. Ethylenedianrillc was llsed as the cathode solution (top) and phosphoric acid as the anode solution (bottom ). The contents of the colunn were pumped ont wit11 a11 LKB l’erpex Peristaltic pump and 0%1111 fractions were collected. The plI of the Ilrldilrttcd fractions was determined at 4°C with a 13cv*kl11:trr b2odel i(i I)igital p1-I meter. .\c~ryl:unide gel elertrofocusing studies were carricstl out rising 1’ ( ampholine in the pH range of 5 8 with T.S’, acrylamide gel. A 25.~1 (860 rg prolc.in ) sample of sheep liver 100,OOOg supernat,:lllt was nlixed with acrylamide-bisacrylamide solrrt ion , and photopolymerized in the presence of riboflavin (17). Cllrrent was applied at 75 V for L’O III. at 4°C:. I~;t,hylenediamine, 1“; (v/v), was IISWI as the cathode solution (top) and 1.25ci phosphoric acid as thra anode solut,ion (bottom). After tllis thr gel was frozen and cut into l-mm slices. I,::rch slice was transferred to a small test t rrhe containing 0.2 ml of water and extracted for IGI hr at 0°C. The pI1 was determined at 4°C using a thill combination electrode. Each tube with the gel slice still present was subjected to glutathioneS-arckricx oxide transferase assay lising 3 /Imoles of t~a~~httl:dt=rre~l ,%-oxide or styrene oxide instead assay, and inof O.(i ~molr as in the standard cllt):lt~~~tl for 20 mill at roon1 temperature (22%).
.)‘),,.I
TIZANSFE;l’,ASP; These were not the best kinetic were used to increase sensitivity parative stlldy. 1: I’SULTS
ANI)
conditions for this
but, corn-
L)ISCUSSION
Two mcathods for mctasuring glutathionc transfwaw activity \\-(w considcwd (13 1; a spt~ctrophotomctric: assay bawd on the increw of absorbance at 330 1~11 upon glutathiow conjugat’ion with 1 ,2-~poxy-:<(in-rlitroph(~nox~~ propaw, :trd :L caolorimrtric assay bawd on the mcasuwmt~nt of the dccrwscb in ~oticclnt,ratioIl of th(b sulfhydryl groups of glutathione as dt>twmincd by thr waction with 5 ,.5’-dithiobis (2.nitrobcnzoatr). Both assays arch rapid and simplr but inscwGtivc1 and thcl former is limitc~tl to a single alkyl clxpoxidc substratcb. A4now radioassay was dcvcIopcd bawd on the formation of radioactive glutathiotw conjugate from naphthalrtwl ,L’-oxidt~ and [“%]glutathioncl (Fig. 1). liecowry of thcl thcl proccdurcb \\-as conj ugat (L through chcckrd by using a standard [“%]glut:~naphthalsw-1 ,2thiow cotijugatch with oxide Lvhich had hcen sgnthwixtd mzy,1 certain amount (front Z-11 matically. nmolcs) of th(x [Y3]gluthathiow conjugate, synthcsixcd as mentioned above, KM added to a reaction mixture which was twutcd as :I st,andard assa\-. Tlw radioactivity \vhicah \\-as rwovcwd ~-as cwmparc~d \vi th the> original radioactivity. Rwowricw avctragcd 52.5 * 0.0 “i. :lssx~~ valurs prrwntc~d ill this papor wrc not corrcrtod for rcwwcyy. The ticn- assay is quite: swsitiw (3w1 though th(a rcwwwy of product is SOI~W\\-hat low. An important advantagc~ is that t#ho assay is applicabh~ to most a1lq.I qoxides and awnc oxides. ‘l’hc~ aesw\- has t h(> advantage of giving clualitutiw c~onfirnlntion of thtl formation of conjugate on p:tp~ chromatograms prior to c>lution and radioactive mcasur(wwt (E‘ig. ‘2). Thcx appropriatc spot,s on chromat~ograms wro identific>d as the glutathiotw conjugatcl \vith r~phthal(w-1 ,L’-osidcl by thrlir positiw wwtion wit’h l<~Cr~O~-AgSO:~ wagc~nt~(IS), by tAicGr absorption of’ short-\~LLvc~lctlgtl-I uv light and by thAr wac+ion nith ninhydrin. Th(l variat,ioti :+ctivit\. of transfrraw with pH is shon-11 in Fig. 3. Thr~ cwzymat,ic wact,ion VW maximal at about S.(i. Ho\v-
226
HAYAKAWA,
Tl2.-
?
I
’
E
I
I
LEMAHIEU, I
I _
_ IO-
AND UDENFRIEND
yielded about 2000 cpm above a blank of about 1500 cpm. Even at 22°C conjugate formation was not linear for more than 5 min, as shown in Fig. 5. In order to maintain initial rates a 5-min incubation period was used in the assay. The effects of glutathione concentration on both the enzymatic and the nonenzymatic formation of conjugate between glutathione and naphthalenc-1,2-oxide are shown in Fig. 6. Conjugate formation in the presence of enzyme exhibited the typical saturation kinetics of an enzyme-catalyzed reaction. The nonenzymatic formation of
PH
curves of gluFIG. 3. (A) The pH activity tathione conjugate formation with naphthaleneenzymatic (47 pg of a CM1,2-oxide. -•---: cellulose column fraction of the sheep liver transferase) and -C--: nonenzymatic. In this and subsequent figures values from the enzymatic and nonenzymatic reactions were all corrected for the blank obtained in the absence of naphthalene1,2-oxide. The enzymatic formation of conjugate was further corrected to obtain the net value by subtracting the value from the nonenzymatic formation of conjugate. Details of the assay conditions are described under Methods. TIME 20-
0
(min)
5. Time course of the formation of glutathione conjugate with naphthalene-1,2-oxide at room temperature (22°C). --•---: enzymatic (107 kg of a DEAE-cellulose column of sheep liver transferase) and -C---: nonenzymatic.
IO
20
30
pg PROTEIN FIG. 4. Transferase activity as a function of protein concentration. A Sephadex G-75 column fraction of sheep liver transferase was used with 402,800 epm of [%]glutathione.
ever, the nonenzymatic reaction increased markedly above pH 8. Incubations were carried out at pH 8 to minimize the nonenzymatic reaction. Conjugate formation as a function of enzyme concentration is shown in Fig. 4. Activity of the order of 2-3 nmoles of conjugate formed during 5 min of incubation
0
2 GSH
I I I I 4 6 CONCENTRATION
I 8
I IO (mM)
6. Effects of glutathione concentration on glutathione conjugate formation with naph-•-: enzymatic (22.8 thalene-1,2-oxide. rg of Sephadex G-75 column fraction of sheep liver transferase) and -C--: nonenzymatic. FIG.
GSH-S-ARENE
OXIDE
conjugate required much higher concentrations of glutathione and exhibited secondorder reaction kinetics, bypical of a pure chemical reaction. Enzymatic activit’y was shown to be destroyed by tryptic digestion and the tryptic effect could be abolished by the addition of soybean trypsin inhibitor. Since significant conjugation between glutathione and arene oxides or alkyl epoxides can proceed nonenzymatically, it was necessary to SW whether nonspecific protcins could catalyze the react’ion. To invcst’igate this, 0.5 mg each of bovine serum albumin, aldolase, L-glutamic acid dehydrogenase, a-globulin, and fibrinogen wre examined for t’ransferase act’ivity. Sorw of these proteins exhibited activity above the controls. The transferase activity was found mainly in liwr and kidney of adult rats, but was also present in many other organs, including skeletal muscle and skin (Table I). Transfcrase activities in liver 100,OOOg suprrnatants from various other species TABLE
I
DISTRIBUTION OF GLUTATHIONE-S-ARENE OXIDE TRANSFERASE ACTIVITY IN VARIOUS RAT TISSUES Organ” Activity (nmoles conjugate formed/g wet wt/min) Cerebrum Cerebellum Brain stem Lung Heart Liver Kidney Spleen Skeletal muscle Skin Small intestine Stomach -
31 34 36 37 19 387 191 30 16 9b 30 30
a Tissues of two adult male Sprague-Dawley rats weighing 500 g each were combined for the experiment. Naphthalene-1 , Z-oxide was used as substrate. b A 0.4.ml aliquot of l~,~Og supernatant from 25% skin homogenate showed 3200 cpm above a blank of about 2500 cpm. All other tissues gave proportionally higher radioactivity.
WY
TRANSFERAHI~: TABLE
II
GLUTATHIONE-S-ARENE OXIDE TR.INSFE;R.\~IC :'uJTIVITIESINLIVER 100,000g SUPERN.ITANTS FROM V.~MOUS SPECIES" Species
Activities (nmoles conjugate formed/g wet wt {min) _~ .-__ With With styrene ouidt naphthalene1,2-oxide
Sheep Horse (frozen) Cattle Hog Human (male, autopsy) Monkey (frozen) Rabbit (frozen) Guinea pig Rat (male) Rat (female) Mouse (male) Mouse (female)
96 152 438 228 187 665 327
f f f f
43 26 94 16
1,562 969 2 2-11 11992
It f Zt *
175 155 217 216
a Details of the preparation of liver IW,OoOg supernatante are described under Methods. b The values represent the mean f 81) oi Four sheep, six male and female Sprague-Dawley rats; five male and female Carworth Farm mice. In all other cases the values represent assays ~)blainrd on tissues from one animal.
were also examined (Table II). An intwcbst ing finding was the significant diffcrcwc bctwcen male and female mice. 1lalc mice showed twice as much activitv as fcwal(l mice with naphthalene-1 , Zoxidc as hub strate; however, with styrene oxidcl as wb strate, there was little diffcrencc br:t\vccn the sexes. Male rats exhibited a somewhat higher activity than female rats lvith both napht’halene-1 , Z-oxide and styrcwb oxide as substrates. The nat’ure of tht>se diffcrorwes is under investigation. All the procedures for t’hc purification4 of glutathione-S-arenc oxide tr:lnsfrrasr act,ivity UYW carried out bet wwl 0” :1nt1 4 Thrl purificat,ion procedure was indt:pt~lttlently developed in this laboratory. W’hilf~ this manuscript was being prepared, a pro~durt~ t’or the purification of rat liver glutathiotle-~‘-alkyl epoxide transferase was made known to us (13) The two procedures were developrd indf~pt~ncl ent,lq- and the similarities are fort,uitorlF
228
HAYAKAWA,
LEMAHIEU,
4°C. Sheep liver supernatant, 500 ml, was adjusted to pH 5.4 with 5 % acetic acid, and the precipitate was removed by centrifugation at 9000 rpm (13,000g) for 45 min in a Sorvall G S rotor. The resultant supernatant was adjusted to pH 7 with 1 M potassium phosphate buffer, pH 7.4, and subjected to ammonium sulfate fractionation. Enzyme activity was precipitated between 20 and 65%, (w/v) of ammonium sulfate. The precipitate was collected by centrifugation and dissolved in a minimal amount of 1 mM potassium phosphate buffer, pH 7.4, and dialyzed for 24 hr against two 4-lit’er changes of the same buffer. The dialyzed fraction (148 ml) was further purified on a DEAE-cellulose column (2.ti X 34 cm) equilibrated with 1 mM potassium phosphate buffer, pH 7.4. The enzyme activity appeared in the void volume on washing with 5 mM phosphate buffer, pH 7.4. The active fractions were combined (140 ml) and dialyzed overnight against 4 liters of Yj mM phosphate buffer, pH 6.5. The dialyzed preparation was next applied to a CR/I-cellulose column (2.5 X 20 cm) which had previously been equilibrated with 5 mM phosphate buffer, pH 6.5. Again the active protein appeared in the void volume on washing with the same buffer. The active fractions (137 ml) were applied to a hydroxylapatite column (2.5 X 9.5 cm) equilTABLE PURIFICATION
OF GLUTATHIONE-S-ARENE
Liver supernatant (100,OOOg) pH 5.4 supernatant Ammonium sulfate fractionation DEAE-cellulose column CM-cellulose column Hydroxylapatite column Sephadex G-75 column Electrofocusingb Peak I Peak II
(20-65yc)
AND
UDENFRIEND
ibrated with 5 mM phosphate buffer, pH 6.5. After washing with 10 mM phosphate buffer, pH 6.5 (ca. 325 ml), the column was subjected to a linear gradient from 10 mM to 100 mM phosphate buffer, pH 6.5 (300 ml in each reservoir). The active protein appeared in the fractions which were 50-60 mM in phosphate concentration. Active fractions from the hydroxylapatite column were pooled and concentrated by ammonium sulfate fractionation ((r65 %), and the precipitate was dissolved in about 7 ml of 1 mM phosphate buffer, pH 7.4. The solution was applied to a Sephadex G-75 column (2.5 X 95 cm) equilibrated with 5 mM phosphate buffer, pH 6.5, containing 0.1 N NaCl, and the column was then washed with the same buffer. Peak fractions were collected and stored in a freezer. The results of the purification of the transferase from sheep liver are summarized in Table III. As a final step, 11 mg protein from the peak fractions of the Sephadex G-75 step were subjected to electrofocusing on a column containing 1% ampholine of pH range 5-8. The results of the electrofocusing are shown in Fig. 7. Enzyme activity separated into several forms, two major peaks and two to three minor peaks, ranging in isoelectric point from pH 6.5 t’o 7.5. Furthermore, activities assayed with both naphthalene-l,2-oxide and styrene oxide as III
OXIDE TR~INSFERME
ACTIVITY
Protein bd
Sp acta
17,400 9,943 6,290 1,848 771 261 101
4.2 5.8 7.0 24.4 45.0 102.0 192.9 292.6 332.0
FROM SHEEP LIVEI~ Yield (%I 100 80 61 tY2 48 37 27
Purification factor 1 1.4 1.7 5.8 11 24 46 70 79
Q With the standard assay using naphthalene-1,2-oxide as substrate and expressed as nmoles of conjugate formed/mg/min. h The activities of the two major peak fractions are shown; Peaks I and II correspond to I and II in Fig. IV.
GSH-S-ARENE
OSII>E
229
TRANSFP:RiZSl~:
4 80C
-460C a 5 0 '-
‘,
0i
3
i 20
40
60
Fractions
80 (0.8
100
120
ml/tube)
FIG. 7. Isoelectric focusing of glutathione-S-arene oxide transferase. Theenzyme protein (11 mg) from the Sephadex G-75 column preparation was applied to the column and r,ln as described in Methods. Peaks I and II correspond to those shown in Table III.
suhstratcs wert~ found to almost parallel ctach other in all the peaks. Further invcstigation of these multiple forms were carried out’ on acrylamide gel clectrofocusing n-it,h crude extracts of sheep liver. Similar multiple forms, two major peaks and two to thrw minor peaks, w-err found in crude c>xtracts. Disc gel electrophoresis of the peak II fraction from the clectrofocusing column, by the method of Ornstcin (19) and Davis (20), rcwaled two major protein bands (about :$O and 50% of total protein by Coomassicl blue staining) which ran close together. It’ was shown that the protein bands were catalytically active, but it was not possihlc to sholz- which of the two prot&l bands on the gcbl was active. Furhher purification is in progrcw to obtain a homogeneous preparation. Thfl molrcular wcxight of th(t partially tw-ific>d transf(lraw (after the hydroxylapatite column stctp) I\-as determined by using a calibrated Scphadcx G-75 column and wt,imatcld (‘1) as 40,000. This valw is
Substrates
(ilutathione Naphthalene-1,2oxide Styrene oxide
K, values (nm) pH 6.5
pH i
pH X
10 0.08
5
I .ti 0.1 I 0. 1:3
R K, values for glut,athione were determlned with a naphthalene-1,2-oxide concentration of 0.6 mM. K,, values for naphthalene-1,2-oxide and styrene oxide were determined using 2-tritiated or 7-tritiated styrtrne naphthalene-1,2-oxide oxide, and nonradioactive glutathionr as slit)strates. The glutathione concentjration was 5 111~. Details of the assay conditions are desc&ribed urrde~ Methods.
230
HAYAKAWA,
LEMAHIEU,
Enzyme preparations after Sephadex gel filtration were fairly stable on storage at -90°C. They gradually lost activity over a period of a few months. The effect of divalent cations on transferase activity was examined and it was found that 2 mM CaC12, MgC&, and ZnCL, neither activated nor inhibited. However, 2 mM CuSO4 caused 73% inhibition. Transferase activity was not inhibited by 2 mM EDTA and was not activated by 0.1 mlvI FAD, pyridoxal phosphate, or thiamine pyrophosphate. K, values for both glutathione and naphthalene-1,2-oxide, determined at different pH values, are summarized in Table IV. K, values for glutathione decreased with increasing pH, whereas the K, values for naphthalene-1,2-oxide were the same at two different pH values, 6.5 and 8. Similar results have been reported in the case of the rat liver enzyme (13). The K, value for glutathione at pH 8 was calculated as 1.6 mM; however, for the standard assay system 0.5 mM glutathione was used to increase the sensitivity of the assay and also t’o minimize the nonenzymatic formation of conjugate. Sheep liver transferase is highly specific with respect to glutathione; however, the enzyme accepts many different oxides as substrates. Details of substrate specificity and structure-activity relationships of a large number of arene oxides and alkyl epoxides will be discussed in a subsequent communication (22). Recently, one of the purest preparations of rat liver glutathione-S-alkyl epoxide transferase obtained by Fjellstedt et al. (13) was supplied to us by Dr. W. B. Jakoby and was found to contain comparable amounts of transferase activity, using the new sensitivc assay system with naphthalene-1 ,2oxide as substrate. It appears now that the failure to observe arene oxide activity in their preparations (13) was due to a lack of sensitivity and reproducibility of the method used to detect arene oxide-glutathione conjugates.5 5 Personal communication Jakoby, National Institutes MD.
from Dr. W. B. of Health, Bethesda,
AND
UDENFRIEND ACKNOWLEDGMENTS
We acknowledge the help and advice of Dr. Kenneth Gibson with the work involving electrofocusing. REFERENCES 1. HAYAKAWA, T. (1973) Fed. Proc. 32, 666. 2. LEIBMAN, K. C., AND ORTIZ, E. (1970) J. Pharmacol. Exp. Ther. 173, 242. 3. JERINA, D. M., DALY, J. W., WITKOP, B., ZALTZMAN-NIRENBERG, P., AND UDENFRIEND, S. (1970) Biochemistry 9, 147. 4. MAYNERT, E. W., FOREMAN, R. L., AND WATABE, T. (1970) J. Biol. Chem. 246, 5234. 5. JERINA, D. M., DALY, J. W., WITKOP, B., ZALTZMAN-NIRENBERG, P., AND UDENFRIEND, S. (1968) Arch. Biochem. Biophys. 128, 176. 6. JERINA, D. M., ZIFFER, M., AND DALY, J. W. (1970) J. Amer. Chem. Sot. 92, 1056. 7. OESCH, F., JERINA, D. M., AND DALY, J. W. (1971) Arch. Biochem. Biophys. 144, 253. 8. BOYLAND, E., AND WILLIAMS, K. (1965) Biothem. J. 94, 190. 9. GROVER, P. L., HEWER, A., AND SIMS, P. (1973) Fed. Eur. Biochem. Sot. Lett. 34, 63. 10. GROVER, P. L., AND SIMS, P. (1970) Biochem. Pharmacol. 19, 2251. 11. GROVER, P. L., FORRESTER, J. A., AND SIMS, P. (1970) Biochem. Pharmacol. 20, 1297. 12. KUROKI, T., AND HEIDELBERGER, C. (1972) Biochemistry 11, 2116. 13. FJELLSTEDT, T. A., ALLEN, R. H., DUNCAN, B. K., AND JAKOBY, W. B. (1973) J. Biol. Chem. 248, 3702. 14. VOGEL, E., AND KLXRNER, F. G. (1968) Angezu. Chem. Int. Ed. Engl. 7, 374. 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND R.aNDALL, R. J. (1951) J. Biol. Chem. 193, 265. 16. BOHLEN, P., STEIN, S., DAIRMAN, W., AND UDENFRIEND, S. (1973) Arch. Biochem. Biophys. 166, 213. 36, 17. WRIGLEY, C. W. (1968) J. Chromatogr. 362. 18. KNIGHT, R. H., AND YOUNG, L. (1958) Biothem. J. 70, 111. 19. ORNSTEIN, L. (1964) Ann. N. Y. Acad. Sci. 121, 321. 20. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404. 21. DETERMANN, H. (1968) Gel Chromatography, 2nd ed., p. 110, Springer-Verlag, New York. 22. HAYAKAWA, T., UDENFRIEND, S., YAGI, H., AND JERINA, D. M., In preparation.