a new class of rat glutathione s-transferase yrs-yrs inactivating reactive sulfate esters as...

10
THE JOURNAL OF BIOI,OGICA~ CHEX.SSTRY @ 1990 by The American Society for Biochemistry and MolecuIar Biology, Inc. Vol. 265, No. 20, 188~ of July 15, pp. 11973-11981, 1990 Printed m U.S. A. A New Class of Rat Glutathione s-Transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols* (Received for publication, October 10, 1989) Akira Hiratsuka, Noriyuki Sebata, Kouske Kawashima, Haruhiro Okuda, Kenichiro Ogura, Tadashi Watabe$& Kimihiko SatohV, Ichiro Hatayamaq, Shigeki Tsuchidaq, Takashi Ishikawall, and Kiyomi Satoll From the $Laboratoty of Drug Metabolism & Toxicology, Department of Hygienic Chemistry, Tokyo College of Pharmacy, Hachioii-shi, Tokyo 192-03, Jawn and the Wecond DeDartment of Biochemistry, Hirosaki University School of Medicine, .&if&ho, fiirosaki 036, Japai A glutathione (GSH) ,S-transferase (GST), catalyzing the inactivation of reactive sulfate esters as metabo- lites of carcinogenic arylmethanols, was isolated from the male Sprague-Dawley rat liver cytosol and purified to homogeneity in 12% yield with a purification factor of 901-fold. The purified GST was a home-dimeric enzyme protein with subunit Mr 26,000 and ~17.9 and designated as Yrs-Yrs because of its enzyme activity toward “reactive sulfate esters.” GST Yrs-Yrs could neither be retained on the ELhexylglutathione gel col- umn nor showed any activity toward 1,2-dichloro-4- nitrobenzene, 4-nitrobenzyl chloride, and 1,2-epoxy- 3-(4’-nitrophenoxy)propane. l-Chloro-2,4-dinitro- benzene was a very poor substrate for this GST. l- Menaphthyl sulfate was the best substrate for GST Yrs-Yrs among the examined mutagenic arylmethyl sulfates. The enzyme had higher activities toward ethacrynic acid and cumene hydroperoxide. N-termi- nal amino acid sequence of subunit Yrs, analyzed up to the 25th amino acid, had no homology with any of the known class alpha, mu, and pi enzymes of the Sprague- Dawley rat. Anti-Yrs-IgG raised against GST Yrs-Yrs showed no cross-reactivity with any of subunits Ya, Yc, Ybl, Yb2, and Yp. Anti-IgGs raised against Ya, Yc, Ybl, Yb2, and Yp also showed no cross-reactivity with GST Yrs-Yrs. The purified enzyme proved to differ evidently from the 12 known cytosolic GSTs in various tissues of the rat in all respects. Immunoblot analysis of various tissue cytosols of the male rat in- dicated that apparent concentrations of the GST Yrs- Yrs protein were in order of liver > testis > adrenal > kidney > lung > brain > skeletal muscle = heart = small intestine = spleen = skin = 0. In spite of their very weak carcinogenicity, benz[u]anthra- cene (BA)’ and chrysene (CR) turn into extremely potent * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 To whom correspondence should be addressed. ‘The abbreviations used are: BA, benz[u]anthracene; CR, chrysene; 5-HCR, 5-hydroxymethylchrysene; 7-HMBA, 7-hydroxy- methyl-12-methylbenz[u]anthracene;DHBA, 7,12-dihydroxymethyl- benz[u]anthracene; CDNB, l-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene; EPNP, 1,2-epoxy-3-(4’-nitrophen- oxy)propane; GSH, reduced glutathione; GSSG, oxidized glutathione; S-hexyl-SG, S-hexylglutathione; GST, glutathione S-transferase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electropho- resis; hplc, high performance liquid chromatography. carcinogens on methylation at the L-region (7- and 12-posi- tions) for the former (l-3) and at the 5-position for the latter (4,5). In hepatic microsomes from untreated rats, enzymatic oxidation of the methylarenes takes place more readily at their methyl carbons than at the arene moieties, leading to the formation of reactive arene oxides (6, 7), to yield the corresponding hydroxymethylarenes as major metabolites (6- 10) which are also potent carcinogens comparable to the mother compounds (1, 2, 5, 11). The carcinogens, 7-hydroxy- methyl-BA, 7-hydroxymethyl-12-methyl-BA (7-HMBA), 12- hydroxymethyl-7-methyl-BA (12-HMBA), 7,12-dihydroxy- methyl-BA (DHBA), and 5-hydroxymethylchrysene (5-HCR) formed from the corresponding methylarenes by hepatic mi- crosomal monooxygenases are metabolized further in hepatic cytosol fortified with 3’-phosphoadenosine 5’-phosphosulfate, a cofactor for sulfotransferases, to give highly reactive sulfate esters, Ar-CHZ-OSO;, except for 5-HCR sulfate with consid- erable stability (l2), all of which have been isolated from incubation mixtures, identified with authentic specimens (12- 15), and demonstrated to have potent intrinsic mutagenicity toward Salmonellu typhimurium TA 98 (12-15, 17). As to 7-HMBA, DHBA, and 5-HCR, the metabolically formed sulfate esters react selectively and readily with the exocyclic amino groups of the purine bases of calf thymus DNA through their methylene carbon with loss of a sulfate anion as a leaving group (16, 18, 19). The rat liver cytosolic sulfotransferases that activate these carcinogenic hydroxy- methylarenes have recently been identified as hydroxysteroid sulfotransferases (16, 20). The same purine base adducts as mentioned above with respects to 7-HMBA were isolated later by Surh et al. (21) from hepatic DNA of new born rats given the same carcinogen. However, in adult rats, liver has never been reported to be a target organ for hydroxymethylarenes, whereas they show very potent carcinogenicity to the skin of the animals (2). In the adult rat liver cytosol, the metabolically formed sulfate esters of 5-HCR, 7-HMBA, and DHBA are enzymat- ically scavenged so readily by glutathione (GSH) (17, 22, 23) that they cannot exert mutagenicity (17, 22, 23), nor bind covalently to calf thymus DNA (16, 23). From the hepatic cytosolic incubation mixtures fortified with 3’-phosphoaden- osine 5’-phosphosulfate and GSH, stable and nonmutagenic S-(aryl)methylglutathiones, Ar-CH*-SG, have been isolated and identified with authentic specimens (17, 22, 23). These facts, therefore, indicate that GST does play an important role in preventing rat liver, having the highest level of hy- droxysteroid sulfotransferase activity among various tissues (24), from tumorigenesis caused by the carcinogens, methyl- and hydroxymethylarenes. The present paper deals with (i) 11973 by guest on July 27, 2015 http://www.jbc.org/ Downloaded from

Upload: magdainfante

Post on 05-Dec-2015

221 views

Category:

Documents


1 download

DESCRIPTION

this an article private, and here is free

TRANSCRIPT

Page 1: A New Class of Rat Glutathione S-transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols

THE JOURNAL OF BIOI,OGICA~ CHEX.SSTRY @ 1990 by The American Society for Biochemistry and MolecuIar Biology, Inc.

Vol. 265, No. 20, 188~ of July 15, pp. 11973-11981, 1990 Printed m U.S. A.

A New Class of Rat Glutathione s-Transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols*

(Received for publication, October 10, 1989)

Akira Hiratsuka, Noriyuki Sebata, Kouske Kawashima, Haruhiro Okuda, Kenichiro Ogura, Tadashi Watabe$& Kimihiko SatohV, Ichiro Hatayamaq, Shigeki Tsuchidaq, Takashi Ishikawall, and Kiyomi Satoll From the $Laboratoty of Drug Metabolism & Toxicology, Department of Hygienic Chemistry, Tokyo College of Pharmacy, Hachioii-shi, Tokyo 192-03, Jawn and the Wecond DeDartment of Biochemistry, Hirosaki University School of Medicine, .&if&ho, fiirosaki 036, Japai

A glutathione (GSH) ,S-transferase (GST), catalyzing the inactivation of reactive sulfate esters as metabo- lites of carcinogenic arylmethanols, was isolated from the male Sprague-Dawley rat liver cytosol and purified to homogeneity in 12% yield with a purification factor of 901-fold. The purified GST was a home-dimeric enzyme protein with subunit Mr 26,000 and ~17.9 and designated as Yrs-Yrs because of its enzyme activity toward “reactive sulfate esters.” GST Yrs-Yrs could neither be retained on the ELhexylglutathione gel col- umn nor showed any activity toward 1,2-dichloro-4- nitrobenzene, 4-nitrobenzyl chloride, and 1,2-epoxy- 3-(4’-nitrophenoxy)propane. l-Chloro-2,4-dinitro- benzene was a very poor substrate for this GST. l- Menaphthyl sulfate was the best substrate for GST Yrs-Yrs among the examined mutagenic arylmethyl sulfates. The enzyme had higher activities toward ethacrynic acid and cumene hydroperoxide. N-termi- nal amino acid sequence of subunit Yrs, analyzed up to the 25th amino acid, had no homology with any of the known class alpha, mu, and pi enzymes of the Sprague- Dawley rat. Anti-Yrs-IgG raised against GST Yrs-Yrs showed no cross-reactivity with any of subunits Ya, Yc, Ybl, Yb2, and Yp. Anti-IgGs raised against Ya, Yc, Ybl, Yb2, and Yp also showed no cross-reactivity with GST Yrs-Yrs. The purified enzyme proved to differ evidently from the 12 known cytosolic GSTs in various tissues of the rat in all respects. Immunoblot analysis of various tissue cytosols of the male rat in- dicated that apparent concentrations of the GST Yrs- Yrs protein were in order of liver > testis > adrenal > kidney > lung > brain > skeletal muscle = heart = small intestine = spleen = skin = 0.

In spite of their very weak carcinogenicity, benz[u]anthra- cene (BA)’ and chrysene (CR) turn into extremely potent

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 To whom correspondence should be addressed. ‘The abbreviations used are: BA, benz[u]anthracene; CR,

chrysene; 5-HCR, 5-hydroxymethylchrysene; 7-HMBA, 7-hydroxy- methyl-12-methylbenz[u]anthracene;DHBA, 7,12-dihydroxymethyl- benz[u]anthracene; CDNB, l-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene; EPNP, 1,2-epoxy-3-(4’-nitrophen- oxy)propane; GSH, reduced glutathione; GSSG, oxidized glutathione; S-hexyl-SG, S-hexylglutathione; GST, glutathione S-transferase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electropho- resis; hplc, high performance liquid chromatography.

carcinogens on methylation at the L-region (7- and 12-posi- tions) for the former (l-3) and at the 5-position for the latter (4,5). In hepatic microsomes from untreated rats, enzymatic oxidation of the methylarenes takes place more readily at their methyl carbons than at the arene moieties, leading to the formation of reactive arene oxides (6, 7), to yield the corresponding hydroxymethylarenes as major metabolites (6- 10) which are also potent carcinogens comparable to the mother compounds (1, 2, 5, 11). The carcinogens, 7-hydroxy- methyl-BA, 7-hydroxymethyl-12-methyl-BA (7-HMBA), 12- hydroxymethyl-7-methyl-BA (12-HMBA), 7,12-dihydroxy- methyl-BA (DHBA), and 5-hydroxymethylchrysene (5-HCR) formed from the corresponding methylarenes by hepatic mi- crosomal monooxygenases are metabolized further in hepatic cytosol fortified with 3’-phosphoadenosine 5’-phosphosulfate, a cofactor for sulfotransferases, to give highly reactive sulfate esters, Ar-CHZ-OSO;, except for 5-HCR sulfate with consid- erable stability (l2), all of which have been isolated from incubation mixtures, identified with authentic specimens (12- 15), and demonstrated to have potent intrinsic mutagenicity toward Salmonellu typhimurium TA 98 (12-15, 17).

As to 7-HMBA, DHBA, and 5-HCR, the metabolically formed sulfate esters react selectively and readily with the exocyclic amino groups of the purine bases of calf thymus DNA through their methylene carbon with loss of a sulfate anion as a leaving group (16, 18, 19). The rat liver cytosolic sulfotransferases that activate these carcinogenic hydroxy- methylarenes have recently been identified as hydroxysteroid sulfotransferases (16, 20). The same purine base adducts as mentioned above with respects to 7-HMBA were isolated later by Surh et al. (21) from hepatic DNA of new born rats given the same carcinogen. However, in adult rats, liver has never been reported to be a target organ for hydroxymethylarenes, whereas they show very potent carcinogenicity to the skin of the animals (2).

In the adult rat liver cytosol, the metabolically formed sulfate esters of 5-HCR, 7-HMBA, and DHBA are enzymat- ically scavenged so readily by glutathione (GSH) (17, 22, 23) that they cannot exert mutagenicity (17, 22, 23), nor bind covalently to calf thymus DNA (16, 23). From the hepatic cytosolic incubation mixtures fortified with 3’-phosphoaden- osine 5’-phosphosulfate and GSH, stable and nonmutagenic S-(aryl)methylglutathiones, Ar-CH*-SG, have been isolated and identified with authentic specimens (17, 22, 23). These facts, therefore, indicate that GST does play an important role in preventing rat liver, having the highest level of hy- droxysteroid sulfotransferase activity among various tissues (24), from tumorigenesis caused by the carcinogens, methyl- and hydroxymethylarenes. The present paper deals with (i)

11973

by guest on July 27, 2015http://w

ww

.jbc.org/D

ownloaded from

Page 2: A New Class of Rat Glutathione S-transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols

11974 Glutathione S-transferase Yrs- Yrs

the existence of at least three GSTs in the rat liver cytosol, which catalyzed GSH conjugation of reactive sulfate esters of carcinogenic arylmethanols and were all unretainable on an S-hexyl-SG affinity column, (ii) the isolation and purification of the major one of these GSTs, which was designated as Yrs- Yrs (a homo-dimeric Y protein that catalyzes GSH conjuga- tion of the “reactive sulfate esters”) and had no homology in N-terminal amino acid sequence to any of the known classes of rat GST isozymes, (iii) the unique properties of GST Yrs- Yrs in substrate specificity and immunochemical reactivity, and (iv) the tissue distribution of GST Yrs-Yrs in the rat, estimated by immunoblot analysis.

EXPERIMENTAL PROCEDURES AND RESULTS*

Purification and Molecular Significance of Rat Liver Cyto- solic G’ST for Inactivating Arylmethyl Sulfates-A preliminary study carried out by using rat liver cytosol prior to establishing a purification scheme for GST, tentatively designated as RS, catalyzing the GSH conjugation of 5HCR sulfate, the most stable compound among highly mutagenic arylmethyl sul- fates, provided two important facts; one was that, unlike rat liver soluble GSTs with subunit proteins Ya, Ybl, YbZ, Ye, and Yp, GST RS had little affinity for and, consequently, passed through an S-hexyl-SG-labeled Sepharose 6B column, and the other that the enzyme had strong affinity for the sulfonated azo dye-labeled gel (blue Sepharose) column. On direct application of the rat liver cytosol to the S-hexyl-SG- gel column, approximately 96% of the hepatic cytosolic activ- ity for GSH conjugation of 5HCR sulfate passed through the column, whereas 86% of the cytosolic activity for GSH con- jugation of I-chloro-2,4-dinitrobenzene (CDNB) was retained on this column. Thus, the S-hexyl-SG-labeled affinity column could be used for elimination of the known GST subunit proteins interacting with this column from the rat liver cy- tosol, and the sulfonated azo dye-labeled column for adsorp- tion of the GST active toward arylmethyl sulfates during the course of purification.

For isolation of GST RS, the rat liver cytosol was applied to an anion-exchange gel (DE-52) column. Most of the cyto- solic RS, surveyed by the assay of the 5HCR sulfate-GSH- conjugating activity, was retained at pH 8.25 on this column and then eluted with NaCl (Fig. 1 in the Miniprint). By this column chromatographic procedure, more than 90% of non- RS proteins were removed. From the RS fraction (Fraction 120-140) of the anion-exchange chromatogram, most of non- RS GSTs active toward CDNB were removed by adsorption on the S-hexyl-SG-gel column (Fig. 2 in the Miniprint). The GST RS protein in the flow-through fraction of the S-hexyl- SG-gel column chromatogram was applied to a chromato- focusing gel column to remove non-RS GSTs active toward CDNB completely (Fig. 3 in the Miniprint). The major GST RS peak, corresponding to the chromatofocusing fractions (Fraction 54-63) eluted at pH 8.2-8.0, contained no detectable activity toward CDNB (Fig. 3 in the Miniprint). The major GST RS protein isolated by chromatofocusing was collected on and eluted from the blue Sepharose column (Fig. 4 in the Miniprint) and then subjected to gel filtration hplc carried out on a TSK gel G3000 SW column for further purification to homogeneity (Fig. 5 in the Miniprint).

The purified RS preparation, obtained in 12% yield with a purification fold of 901 (Table I), had an apparent Mr value

* Portions of this paper (including “Experimental Procedures,” part of “Results,” Figs. l-6, and Table II) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

TABLE I Summary of the purification of GST RS Wrs-Yrs)

from rat liver cytosol Liver cytosol from 20 male adult Sprague-Dawley rats was used.

GST Yrs-Yrs was retained on a DE-52 column at an alkaline pH and passed through an S-hexyl-SG affinity column. From the flow- through fraction of the affinity column chromatogram, GST Yrs-Yrs was separated by chromatofocusing and then retained on a blue Sepharose column. The enzyme was purified to homogeneity by hplc on a TSK gel G3000 SW column after being eluted from the blue Sepharose column. The enzyme activity was measured with 5HCR sulfate and protein concentrations by the method of Lowry et al. (27).

Purification step Total Total Specific Purifi- protein activity activity cation ““’

w nmolf nmol/ min min/mg -fold %

Rat liver cytosol 6248 500 0.08 1 100 DE-52 397 380 0.96 12 76 S-Hexyl-SG 371 363 0.98 12 73 Chromatofocusing 20.1 183 9.10 114 37 Blue Sepharose 3.94 143 36.3 454 29 TSK gel G3000 SW 0.86 62 72.1 901 12

of 54,000, estimated from the retention volume on the TSK gel column in a comparative chromatographic study carried out by using the Mr marker proteins, bovine serum albumin (M, 66,000), egg albumin (Mr 45,000), and bovine carbonic anhydrase (Mr 29,000). SDS-polyacrylamide gel electropho- resis of the RS preparation indicated that the M? 54,000 enzyme protein consisted of two identical subunit proteins with an Mr value of 26,000 (Fig. 6 in the Miniprint), estimated from the result of a comparative electrophoretic study carried out on the same gel plate by using not only various Mr marker proteins but the authentic GST subunit proteins, Ya (Mr 25,000), YbI and Yb2 (Mr 26,500), and Yc (28,000), isolated and purified from the rat liver cytosol. Therefore, based on these molecular data, the homo-dimeric enzyme GST RS was re-designated as GST Yrs-Yrs.

GST Yrs-Yrs had a p1 value of 7.9, determined by the isoelectricfocusing method. The N-terminal amino acid se- quence of this enzyme, determined from the N-terminal to 25th amino acids with an automatic amino acid sequencer based on the Edrnan degradation method, was as follows: Gly- Leu-Glu-Leu-Tyr-Leu-Asp-Leu-Leu-Ser-Gln-Pro-Ser-Arg- Ala-Val-Tyr-Ile-Phe-Ala-Lys-Lys-Asn-Gly-Ile-,

A co-hplc study, carried out on an octadecylsilica column developed with aqueous acetonitrile containing 0.1% (v/v) trifluoroacetic acid in gradient manner, indicated that the subunit protein Yrs was eluted as a single peak at a completely different retention time from those of the rat liver cytosolic GST subunits which were collected on and eluted from the S- hexyl-SG affinity column into a single fraction (Fig. 7). Under the hplc conditions, all the dimeric enzymes examined were dissociated into subunits. The subunit Yrs was less polar than GST subunits Ybl, Yb2, and Yc and more polar than Ya, the last one of which was eluted as a doublet peak as had been demonstrated by Ostland Farrants et al. (39). These subunit peaks were all identified with the corresponding pure homo- dimeric GSTs isolated from the rat liver cytosol.

Amino acid composition of an acid hydrolyzate of GST Yrs- Yrs indicated that the enzyme contained Glx (Glu and Gln) in the highest molar ratio and, next to this, Leu, Ala, Gly, and Asx (Asp and Asn) in decreasing order (Table II in the Miniprint).

Substrate Specificities of GST Yrs-Yrs-GST Yrs-Yrs cat- alyzed GSH conjugation of three examined mutagenic, reac- tive sulfate esters of arylmethanols other than 5-HCR sulfate (Table III). Of the four sulfate esters, 1-menaphthyl sulfate

by guest on July 27, 2015http://w

ww

.jbc.org/D

ownloaded from

Page 3: A New Class of Rat Glutathione S-transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols

Glutathione S-transferase Yrs- Yrs

Ya

L L U

FIG. 7. Separation of rat liver cytosolicOGST subunits by reverse phase hplc. A PBondasphere C&300 A column (3.9 mm x 15 cm) was eluted at a flow rate of 1 ml/min with a 35-55% (v/v) linear gradient of acetonitrile in water, both containing 0.1% (v/v) trifluoroacetic acid. A, GST fraction (20 pg of protein) isolated from rat liver cytosol by S-hexyl-SG affinity chromatography as described in the text. B, purified GST Yrs-Yrs (5 pg). C, a mixture of A and B in water. U represents unidentified proteins.

was the best substrate for this GST. A kinetic study, carried out by the double reciprocal plot method based on apparent rates for the enzymatic GSH conjugate formation from 5HCR sulfate (lo-50 PM) at pH 7.4, indicated that purified GST Yrs-Yrs had a K,,, value of 17.7 PM and a !Z~~~ value of 0.123 s-l. A Km value for GSH was 1.34 mM in the 5-HCR sulfate conjugation reaction catalyzed by this enzyme at pH 7.4.

A comparative study carried out by using purified specimens of six major rat liver cytosolic GSTs with high affinity for the S-hexyl-SG-gel column and high activities toward CDNB indicated that only two GSTs bearing subunit protein Yc could catalyze the GSH conjugation reaction of 5-HCR sulfate

TABLE III Substrate specificity of purified GST Yrs- Yrs toward reactive

arylmethyl sulfates GSH conjugation of arylmethyl sulfates was determined by the

rate assay for l-menaphthyl sulfate as reported previously (30) and by direct fluorophotometry of S-(aryl)methylglutathiones formed in the incubation mixtures for the others as described in the text. Rates of nonenzymatic GSH conjugations, obtained by using boiled GST Yrs-Yrs, were subtracted from those of the enzymatic reactions.

Substrate Specific activity

mnol/min/mg l-Menaphthyl sulfate 170.4 7-HMBA sulfate 148.2 5-HCR sulfate 79.2 DHBA 7-sulfate 51.5

TABLE IV GSH conjugations of 5-HCR sulfate and CDNl3 by various GSTs

purified from Sprague-Dawley rat liver cytosol Enzymatic GSH conjugations of 5-HCR sulfate and CDNB were

measured as described in the text. GST 5-HCR sulfate

Ya-Ya Ya-Yc Yc-Yc Ybl-Ybl Ybl-Yb2 Yb2-Yb2 Yrs-Yrs

nmol/minfmg ~mnl/mia/mg ND’ 33.0 0.24 26.0 0.75 17.2

ND 28.3 ND 32.9 ND 19.6 7a.9 0.1

’ ND, not detectable (less than 0.05 nmol/mg protein/min).

TABLE V

Substrate specificity of GST Yrs- Yrs The enzymatic activity toward cumene hydroperoxide was ex-

pressed as GSH peroxidase by determining GSSG formed from GSH. Other substrates were used for measuring GSH conjugation activities of the enzyme. BP, bromosulfophthalein.

Substrate Sue&c activitv

CDNB DCNB EPNP Ethacrynic acid tran.s-4-Phenyl-3-buten-Z-one BP 4.Nitrobenzyl chloride 4.Nitrophenyl acetate l-Menaphthyl sulfate Cumene hydroperoxide

~mol/min/mg SO.1 ND”,b ND’

0.25 0.02

NDb NDd ND*

0.17 1.76

’ ND, not detectable. * Less than 2 nmol/mg protein/min. ’ Less than 10 nmol/mg protein/min. ‘Less than 5 nmol/mg protein/min.

(Table IV). However, their activities toward 5-HCR sulfate was only 0.3 and 1% for GSTs Ya-Yc and Yc-Yc of that exerted by GST Yrs-Yrs. Other rat liver GSTs bearing the subunit proteins Ybl and Yb2 and GST Ya-Ya had no activity toward 5-HCR sulfate, whereas all of these GSTs had high activities toward CDNB that was an extremely poor substrate for GST Yrs-Yrs.

Representative GST substrates were examined for GSH conjugations by GST Yrs-Yrs (Table V). None of DCNB, EPNP, BSP, 4-nitrobenzyl chloride, and 4-nitrobenzyl acetate was a substrate for this enzyme. Ethacrynic acid was a better substrate than l-menaphthyl sulfate. Cumene hydro- peroxide was reduced at a considerable rate by this enzyme

by guest on July 27, 2015http://w

ww

.jbc.org/D

ownloaded from

Page 4: A New Class of Rat Glutathione S-transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols

11976 Glutathione S-t)

FIG. 8. Ouchterlony double immunodiffusion. A, the center well contained anti-YrsIIgG raised against GST Yrs-Yrs (Abl). WeUs 1 (GST Yrs-Yrs), 2 (GST Ya-Yc), 3 (GST Ybl-Yb2), and 4 (GST Yu-YD) contained 6 ug each of respective enzyme proteins puritied from rat liver cytosol. g, well 1 contained 6 pg of GST Yrs-Yrs. Wells Abl (against GST Yrs-Yrs), Ab2 (against GST Ya-Yc), Ab3 (against GST Ybl-Yb2), and Ab4 (against GST Yp-Yp) contained sufficient amounts of IgG fractions of rabbit antisera. C, weU 5 contained a mixture of 6 pg each of GSTs Ya-Yc, Ybl-Yb2, and Yp-Yp. The welLs Abl-Ab4 contained the same amounts of the anti-IgG fractions as used for B.

with concomitant formation of GSSG from GSH. Immunochemical Properties of GST Yrs- Yrs-An anti-IgG

preparation from rabbit antisera raised against purified GST Yrs-Yrs caused significant precipitation against the antigen, but showed no cross-reactivity with GSTs Ya-Yc, Ybl-Yb2, and Yp-Yp, isolated as homogenous proteins from the rat liver cytosols, when examined by the Ouchterlony double- immunodiffusion test (Fig. 8A). Moreover, anti-IgG prepara- tions from rabbit antisera raised against purified GSTs Ya- Yc, Ybl-Yb2, and Yp-Yp also showed no cross-reactivity with the enzyme GST Yrs-Yrs, whereas under the same conditions, the anti-GST Yrs-Yrs-IgG formed a significant precipitate against the antigen (Fig. 8B). These antibodies as well as that against GST Yrs-Yrs had all sufficient immunoreactivities with the corresponding purified GSTs as antigens (Fig. 8C).

Tissue Distribution of GST Yrs-Yrs in the Rut-Immuno- blot analysis of various tissue cytosols of the male rats, carried out by using SDS-polyacrylamide gel electrophoresis and the anti-GST Yrs-Yrs-IgG preparation, suggested that testis con- tained a high concentration of GST Yrs-Yrs, comparable with that in the liver and also that the enzyme existed at somewhat lower concentrations in adrenal and kidney and at a much lower concentration in lung (Fig. 9). GST Yrs-Yrs, however, existed at extremely low concentrations in all the cytosols of the skin, heart, small intestine, and spleen, so that it could not be detected unless much larger amounts of these cytosolic proteins (at least more than five times of the liver protein) were applied.

pansferase Yrs- Yrs

45,000 +

36,000 +

29,000 +

24,000 -m

20,100 +

45,000 -b

36,000 -w

29,000 -b

24,000 +

20,100 -b

3 4 5 6 7 8

11 12 13 14 15

-- -

FIG. 9. Immunoblot analysis of GST Yrs-Yrs in the cytosols of various tissues of rats. Protein samples were resolved by SDS- polyacrylamide gel electrophoresis (15% gel) and transferred electro- phoretically to a nitrocellulose membrane. The membrane was se- quentially incubated with 3% bovine serum albumin, IgG of rabbit antiserum (at dilution of l/1000) raised against the purified enzyme, goat anti-rabbit IgG, rabbit peroxidase anti-peroxidase, and finally with 50 mM Tris-I-El buffer, pH 7.5, containing hydrogen peroxide and 3.3’-diaminobenzidine as reoorted nreviouslv (38). Lanes 1, 8, 9, and 15, GST Yrs-Yrs (0.05 pg each); lanes 2-5 and 7~ cytosolic proteins (20 fig each) of adrenal, brain, heart, kidney, and lung, respectively; lanes 6 and 14, cytosolic proteins (5 pg each) of liver and testis; lunes 10-14, cytosolic proteins (20 pg each) of skeletal muscle, skin, small intestine, and spleen, respectively. Arrows indicate A4r markers used egg albumin (45,000), rabbit muscle glyceraldehyde-3-phosphate de- hydrogenase (36,000), bovine erythrocyte carbonic anhydrase (29,000), bovine trypsinogen (24,000), and soybean trypsin inhibitor (20,100).

DISCUSSION

So far as concerned with rat liver cytosolic GSTs, little is known of such an isozyme, except GST E (5-5), that is neither retained on the S-hexyl-SG affinity column (40), nor detected by the most widely used standard substrate, CDNB, or/and DCNB (30,41). Extensive studies on the isozymes of GST in the rat liver have been made by using the affinity column and these standard substrates (41), and six major dimeric iso- zymes, GSTs Ya-Ya, Ya-Yc, Yc-Yc, Ybl-Ybl, Ybl-Yb2, and Yb2-Yb2, have been isolated and purified to homogeneity (28, 42). An additional homo-dimeric isozyme, GST P (GST Yp- Yp), was also isolated as a major GST, retainable on the affinity column and detected by CDNB, from the liver cytosol of rats bearing hepatic hyperplastic nodules induced by he- patocarcinogens (29, 43, 44). These seven rat liver GSTs are well studied on their primary structures (45), immunochemi- cal properties (46), substrate specificities, tissue distribution, and species difference (41). Classification of these GSTs has been made in relation to human GSTs a - 6, p, and 7r mainly based on their immunochemical homology and N-terminal amino acid sequences (46). The rat liver GSTs with subunit proteins Ya and Yc belong to class alpha, GSTs with Ybl and Yb2 to class mu, and GST P to class pi (46) (Table VI). N-terminal amino acid sequences of GST subunit proteins in each class have a strong homology and can be readily differ- entiated from those of the other classes of GSTs. As to GST Yrs-Yrs, no homology was found in its N-terminal amino acid sequence and immunochemical property to any of the known rat GSTs. Furthermore, the Yrs subunit protein contained

by guest on July 27, 2015http://w

ww

.jbc.org/D

ownloaded from

Page 5: A New Class of Rat Glutathione S-transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols

Glutathione S-transferase Yrs- Yrs

TABLE VI Comparison of N-terminal amino acid sequences of three classes of rat GST subunits

and Yrs as a new class of GST subunit Underlined N-terminal amino acids of the alpha, mu, and pi classes of enzyme subunits represent the difference

in sequence from those of Yrs.

11977

ChSS GST

subunit“ N-terminal amino acid squerm? Ref.

Alpha

Mu

Pi

Other

YU (1) Yc (2) Yk R-9

Ybl (3) Yb2 (4) Yb3’ (6) Ynl (6) Yn2 (91 Yb4d (?l Yo (11)

YP (71

Yrs (?)

1 5 10 15 20 25 SGKPVLHYFNARGRMECIRWLLAAA-- ----- ------- --- ------- PGKPVLHYFDGRGRMEPIRWLLAAA-- ----- ------- --- ------- EVKPKLYYFqGRGRMEVIRWLLATA-- ----- --- --- --- ------- PMIL --- PMIL --- PMTL --- PMTL --- PVTL --- AMIL --- ?MVL --

LEYT ---- LEYT ---- LEYT ---- L----. Emmmm. ~EYT ---- LEFT ----

DS-- -- DT-- -- DS-- --

,---- ,---- DS-- -- DT-- --

PPYIVYFPVRGRCEATRMLLADQGQ-- -------------- ------- GLELYLDLLSQPSRAVYIFAKKNGI-- 1 5 10 15 20 25

a Numerals in parenthes represent subunit number proposed hy Jakoby et ul. (59). * ?, not assigned. The N-terminal amino acid sequences of the subunit proteins, except Yk, Ynl, Yn2, Yo, and

Yrs, were determined by the cDNA-cloning method. ’ Yb3 has been demonstrated to he identical with Ynl (54). ’ Yb4 exists as a putative subunit encoded merely in Sprague-Dawley rat liver cDNA, and the corresponding

protein has not been detected yet.

higher molar compositions of leucine and histidine residues than that of any other GST (Table II in the Miniprint).

GST Ya, isolated as a minor GST protein from the rat liver cytosol has been demonstrated by an immunochemical study not to belong to class alpha nor to class mu, although no further attempt has been made to classify the isozyme by using the class pi enzyme GST P or an antibody against GST P (47, 48). GST Ya differs from GST Yrs-Yrs in that it can be retained on the S-hexyl-SG affinity column and detected by CDNB with a low activity (much higher than for GST Yrs-Yrs) and has high activities toward EPNP and 4-nitro- phenyl acetate. The N-terminal amino acid sequence has not been shown with this GST isozyme.

Several different isozymes have been also found as major GSTs in the extrahepatic tissues of the rat, all of which had high catalytic activities toward CDNB similarly to the afore- mentioned hepatic GSTs. They were, a class alpha enzyme, GST Yk-Yk, existing in the testis, kidney, and lung (60, 61), and four class mu enzymes, GSTs Ynl-Yn2 and Yn2-Yn2, both in the testis (55), GST Ynl-Ynl in the brain (54, 62), and GST YO-YO in the testis (40, 56). Except GST Yk-Yk, these enzymes were retained on the S-hexyl-SG affinity col- umn (40,54,55,63). In the rat liver cytosol, GST Yk-Yk also existed as a very minor enzyme (41, 61), and the subunit protein Yn as a component of very minor hetero-dimeric GSTs associating with the subunit proteins Ybl and Yb2 (51, 64,65). The class pi enzyme, GST P, exists rather as a major isozyme in the normal rat kidney and small intestine (60, 66, 67). The subunit protein Yk reacted with the antibodies to the class alpha enzyme subunits Ya and Yc, but did not with those of the other classes of enzyme subunits (60). Similarly, the extra-hepatic class mu enzymes reacted only with the antibodies to the hepatic class mu enzyme subunits Ybl and Yb2 (46, 60).

GST Yrs-Yrs isolated in the present study, however, was found to be completely different from any of these three classes of GSTs in all respects so far as estimated from their chromatographic behaviors, immunochemical properties, sub- strate specificities, and N-terminal amino acid sequences (Table VI).

GST Yrs-Yrs was also completely different in substrate specificity and in subunit Mr and p1 values from GST E (5- 5), although both of them appeared in the flow-through frac- tion of the S-hexyl-SG affinity column chromatogram of the rat liver cytosol without being retained. GST E is the only reported GST isozyme that can neither be retained on the affinity column (66), nor show any catalytic activity toward CDNB and DCNB (30,68-70). GST E has been demonstrated to be highly active toward the epoxide EPNP (30, 6%70), toward which GST Yrs-Yrs, however, had no activity, and to be completely inactive toward menaphthyl sulfate (69), the best substrate among examined sulfate esters for GST Yrs- Yrs (Table III). GST E has a subunit Mr value of 24,700 and p1 value of 7.3 with the preparation from Sprague-Dawley rat liver (70) whereas the Mr and p1 values of the hepatic subunit protein Yrs of the Sprague-Dawley rat were 26,000 and 7.9, respectively. Little systematic study has been made on the immunochemical property of GST E, although antibodies raised against a few rat liver GSTs were demonstrated to show no cross-reactivity with this enzyme (30, 71). In addi- tion, no information has been available on the N-terminal amino acid sequence of the GST E protein.

A GST isozyme, designated as 5*-5* because of its similarity to GST 5-5, has very recently been isolated from the nuclei of rat liver cells (72). This enzyme can neither be retained on the S-hexyl-SG affinity column nor cross-reactive with the antibodies against GSTs 1-Z and 3-4. GST 5*-5* has high activities toward EPNP and DNA hydroperoxide, but shows no appreciable activity toward CDNB. The N-terminal amino acid sequence has not been shown with this GST isozyme.

Earlier than 20 years ago, an attempt was made by Gillham (73) to prove the existence of an enzyme catalyzing GSH conjugation of benzyl and menaphthyl sulfates as putative precursors of the benzyl- and menaphthyl-mercapturic acids excreted into the urine of rats given the corresponding aryl- methanols (74,75). He found two GST isozymes active toward menaphthyl sulfate exist in the rat liver cytosol by an isoelec- tric focusing method and partially purified one of them to a purification fold of 76 (76). However, the partially purified

by guest on July 27, 2015http://w

ww

.jbc.org/D

ownloaded from

Page 6: A New Class of Rat Glutathione S-transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols

11978 Glutathiorw S-transferase Yrs- Yrs

GST preparation might be different from GST Yrs-Yrs, be- cause the former was active toward DCNB and 4-nitrobenzyl chloride (69). GST Yrs-Yrs had no appreciable activity toward these chlorinated substrates (Table V). He might have iso- lated one of the two uncharacterized GSTs active toward 5 HCR sulfate other than GST Yrs-Yrs, which were shown in the chromatofocusing chromatogram (Fig. 3 in the Miniprint). Little was reported by Gillham about molecular data of his enzyme. Our preliminary study indicated that the enzyme eluted just behind the Yrs-Yrs peak at a lower pH range in chromatofocusing (Fig. 3 in the Miniprint) was separable from GST Yrs-Yrs on a blue Toyopearl column and had a cross- reactivity with the anti-Yrs-IgG preparation (data not shown).

GSTs play a key role in preventing a variety of reactive metabohtes of xenobiotics from their attacks on cellular biom- acromolecules (41, 77, 78), which may induce necrosis and tumorigenesis of tissues. Either excessive formation or rela- tively low GST-mediated scavenging of the reactive metabo- lites may result in their covalent binding to DNA, leading to mutation or death of cells. A typical example is N-hydroxy- acetylaminofluorene, a hepatocarcinogen, which has been demonstrated to be activated by phenol sulfotransferase IV (79,80) and to be merely scavenged in nonenzymatic manner by GSH (81, 82). However, the active metabolites, sulfate esters of nonhepatocarcinogenic arylmethanols, are rapidly and completely scavenged by GSTs in the rat liver cytosol fortified with GSH (15-17, 22, 23), so that the carcinogens can neither bind covalently to DNA (16, 23) nor induce mutation of cells (15, 17, 22, 23) so far as examined in uitro.

GST Yrs-Yrs may play a central role in scavenging the reactive sulfate esters of carcinogenic arylmethanols in the nontarget organ, rat liver, which has the highest level of hydroxysteroid sulfotransferases activating the carcinogens among all the examined tissues of the rat (24). Rat skin, a well known target organ for 5-HCR (5), which has a sulfo- transferase activity to activate the carcinogen,3 was found by the immunoblotting method to lack in GST Yrs-Yrs. Isolation and purification of the other rat liver GST isozymes active toward 5-HCR sulfate are in progress in our laboratory.

Acknowledgments-We are grateful to Dr. H. Noguchi and K. Noda of Fujisawa pharmaceutical industries Co., Ltd. (Osaka, Japan) for analyses of the N-terminal amino acid sequences and amino acid composition of GST Yrs-Yrs.

REFERENCES

1. Cavalieri, E., Roth, R., Rogan, E., Grandjean, C., and Althoff, J. (1978) in Corcinogene&.s (Jones, P. W., and Freudenthal, R. I., eds) Vol. 3, pp. 273-284, Raven Press, New York

2. Cavalieri, E., Roth, R., and Rogan, E. (1979) in Polynuclear Aromatic Hydrocarbons (Jones, P. W., and Leber, P., eds) pp. 517-529, Ann Arbor Science Publishers, Michigan

3. Selkirk, J. K. (1980) in Carcinogenesis (Slaga, T. J., ed) Vol. 5, pp. l-31, Raven Press, New York

4. Hecht, S. S., Loy, M., and Hoffmann, D. (1976) in Curcinogenesis (Freudenthal, R. I., and Jones, P. W., eds) Vol. 1, pp. 325340, Raven Press, New York

5. Amin, S., Juchatz, A., Furuya, K., and Hecht, S. S. (1981) Curcinogenesis 2, 1027-1032

6. DiGiovanni, J., and Juchau, M. R. (1980) Drug Metab. Reu. 11, 61-101

7. Hecht, S. S., LaVoie, E. J., Mazzarese, R., Amin, S., Bedenko, V., and Hoffmann, D. (1978) Ccmcer Res. 38,2191-2194

8. Dip&e, A. (1976) in ChemicaZ Carcinogens (Searle, C. E., ed) ACS Monograph 173, pp. 245-314, American Chemical Society, Washington, D. C.

‘H. Okuda, M. Hata, H. Nojima, and T. Watabe, unpublished data.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Chou, M. W., Yang, S. K., Sydor, W., and Yang, C. S. (1981) Cuncer Res. 41, 1559-1564

Melikian, A. A., LaVoie, E. J., Hecht, S. S., and Hoffmann, D. (1983) Curciflogenesis 4,843-849

Boyland, E., Sims, P., and Huggins, C. (1965) Nuture 207,816- 817

Okuda, H., Hiratsuka, A., Nojima, H., and Watabe, T. (1986) Biochem. Pharmacol. 35,535-538

Watabe, T., Ishizuka, T., Isobe, M., and Ozawa, N. (1982) Science 215,403-405

Watabe, T., Hiratsuka, A., Ogura, K., and Endoh, K. (1985) Biochem. Biophys. Res. Corn&n. 131,694-699

Watabe. T.. Hakamata. Y.. Hiratsuka. A.. and Ogura. K. (1986) Carcinogenesis 7,207-2i4 -

Watabe, T., Hiratsuka, A., and Ogura, K. (1987) Carcinogenesk 8,445-453

Watabe, T., Hiratsuka, A., and Ogura, K. (1986) Biochem. Bio- phys. Res. Commun. 134,100-105

Watabe. T., Fuiieda. T., Hiratsuka, A.. Ishizuka, T., Hakamata, Y., and C&a, K.‘(l985) Biochem. PharmacoL.34; 3002-3005.

Okuda. H.. Noiima. H.. Miwa. K.. Watanabe. N.. and Watabe. T. (1989) Chem Res. !P&icol. 2, i5-22 ’ ’

Okuda, H., Nojima, H., Watanabe, N., and Watabe, T. (1989) Biochem. Pharmacol. 38,3003-3009

Surh. Y.-J., Lai, C.-C., Miller, J. A., and Miller, E. C. (1987) B&hem. Biophys. Res. Common. 144,576-582

Watabe. T.. Ishizuka. T.. Ozawa. N.. and Isobe. M. (1982)

25. 26.

27.

28.

29.

30.

31.

32. 33.

34. 35.

36.

37.

38.

39.

B&hem. Pharmucol~ 31; 2542-2k44 Okuda, H., Miwa, K., Nojima, H., and Watabe, T. (1986) Biochem.

Phurmncol. 35,4573-4576 Singer, S. S. (1985) in Biochemical Phnrmucology and Toxicology

(gakimi, D., and Vessey, D. A., eds) Vol. 1, pp. 97-159, John Wilev & Sons. Inc.. New York

Clapp,J. J., and Young, L. (1970) Biochem. J. 118,765-771 Vince, R., Daluge, S., and Wadd, W. B. (1971) J. Med. Chem.

14,402-404 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

(1951) J. Biol. Chem. 193, 265-275 Mannervik, B., and Jensson, H. (1982) J. Biol. Chem. 257,9909-

9912 Satoh, K., Kitahara, A., Soma, Y., Inaba, Y., Hatayama, I., and

Sato, K. (1985) Proc. N&l. Acti. Sci. U. S. A. 82, 3964-3968 Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974) J. BioZ.

Chem. 249,7130-7139 Prohaska, J. R., and Ganther, H. E. (1976) J. Neurochem. 27,

1379-1387 Laemmli, U. K. (1970) Nutwe 227,680-685 Penke, B., Ferenczi, R., and Kovacs, K. (1974) Anal. B&hem.

60,45-50 Moore, S. (1963) J. BioZ. Chem. 238, 235-237 Kitahara, A., and Sato, K. (1981) B&hem. Biophys. Res. Com-

mun. 103,943-950 Ouchterlony, 0. (1949) Acta Pathot. Microbial. Scund. 26, 507-

515 Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. N&l. Acud.

Sci. U. S. A. 76,4350-4354 Domin, B. A., Serabjit-Singh, C. J., and Philpot, R. M. (1984)

Anul. Biochem. 136,390-396 Ostlund Farrants, A.-K., Meyer, D. J., Coles, B., Southan, C.,

Aitken, A., Johnson, P. J., and Ketterer, B. (1987) Biochem. J. 245,423-428

40. Hayes, J. D. (1988) Biochem. J. 265,913-922 41. Ketterer, B., Mever, D. J., and Clark, A. G. (1988) in GZutathione

42.

43.

44. 45.

Conjugation, Mechanism and Biological Significance (Sies, H., and Ketterer, B., eds) pp. 74-135, Academic Press, London

Alin, P., Jensson, H., Guthenberg, C., Danielson, U. H., Tahir, M. K.. and Mannervik. B. (1985) Anal. Biochem. 146.313-320

46.

Sato, K:, Kitahara, A., Satoh, K.; Ishikawa, T., Tatematsu, M., and Ito, N. (1984) Gann 75, 199-202

Sato, K. (1988) Jpn. J. Cuncer Res. 79, 556-572 Pickett, C. B., and Lu, A. Y. H. (1988) in Glutathione Conjugation,

Mechanism und Biological Significunce (Sies, H., and Ketterer, B., eds) pp. 138-156, Academic Press, London

Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir, M. K., Warholm, M., and Jornvall, H. (1985) Proc. N&l. Acad. Sci. U. S. A. 82,7202-7206

4?. Reddy, C. C., Li, N.-Q., and Tu, C.-P. D. (1984) Biochem. Biophys. Res. Commun. 121,1014-1020

by guest on July 27, 2015http://w

ww

.jbc.org/D

ownloaded from

Page 7: A New Class of Rat Glutathione S-transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols

Glutathione S- transferase Yrs- Yrs 11979

4%

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60. 61.

62.

63. 64.

Tu, C.-P. D., and Reddy, C. C. (1985) J. BioL. Cnem 260, 9961- 9964

Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argen-

&in, P.; Jensson, H.; Cederlund, E., Jornvall, H., and ~Mannervik,

bright, L.. and Lu A. Y. H. (1984) J. &ol. CIxem. 259, 5182-

B. (1989) B~ocIrem. J. 261,531-539 Ding, G. J.-F., Lu, A, Y. H., and Pickett, C. B. (1985) J. Bial.

5li8

Uzem. 260,13268-13271

Telakowski-Hopkins, C. A., Rodkey, J. A., Bennett, C. D., Lu, A. Y. H.. and Pickett. C. B. (1985) J. BioL Chem. 260.5820-5825

65. Hayes, J. D. (1984) 23~oc/xm. J. 224, 839-852 66. Guthenberg, C., Jensson, H., Nystrom, L., Gsterlund, E., Tahir,

M. K., and Mannervik, B. (1985) &o&em. J. 230,609-615 67. Tahir. M. K.. &er. N., and Mannervik, B. (1988) B~oc~m. J.

Ding, G. J.-F., Ding, V. D.-H., Rodkey, J. A., Bennett, C. D., Lu, A. Y. H., and Pickett, C. B. (1986) J. BioL Chem. 261, 7952- 7957

69. Pabst, M. J., Habig, W. H., and Jakoby, W. B. (1973) Biochem. Biophys. Res. Common. 52,1123-1128

70. Fjellstedt, T. A., Allen, R. H., Duncan, B. K., and Jakoby, W. B.

25$759-i64

(1973) J. Biol. Chem. 248, 3702-3707 71. Jakoby, W. B. (1978) Adv. Enzymol. 46,383-414

68. Meyer, D. J., Christodoulides, L. G., Tan, K. H., and Ketterer,

72. Tan, K. H., Meyer, D. J., Gillies, N., and Ketterer, B. (1988) Biochem. J. 254,841-845

B. (1984) FE&S Z&r. 327-330

Abramovitz, M., and Listowsky, I. (1987) J. Biol. Ckm. 262, 7770-7773

Ishikawa, T., Tsuchida, S., Satoh, K., and Sate, K. (1988) Eur. J. Biochem. 176,551-557

Suguoka, Y., Kano, T., Okuda, A., Sakai, M., Kitagawa, T., and

Kispert, A., Meyer, D. J., Lalor, E., Coles, B., and Ketterer, B.

Muramatsu, M. (1985) Nucleic Acids. Res. 13, 6049-6057 Jakoby, W. B., Ketterer, B., and Mannervik, B. (1984) Biochem.

(1989) Biochem. J. 260, 789-793

Pharmacol. 33, 2539-2540

Lai, H.-C. J., Qian, B., Grove, G., and Tu, C.-P. D. (1988) J. BioZ.

Hayes, J. D., and Mantle, T. J. (1986) BiocheSm. J. 233, 779-788 Meyer, D. J., Lalor, E., Coles, B., Kispert, A., Alin, P., Mannervik,

C&m. 263,11389-11395

B., and Ketterer, B. (1989) Biochem. J. 260, 785-788 Tsuchida, S., Izumi, T., Shimizu, T., Ishikawa, T., Hatayama, I.,

Satoh, K., and Sate, K. (1987) Eur. J. B&hem. 170, 159-164 Hayes, J, D. (1986) Biochem. J. 233, 789-798 Hayes, J. D., and Chalmers, J. (1983) Biochem. J. 215, 581-588

76. Gillham, B. (1973) Biochem. J. 135,797-804 77. Chasseaud, L. F. (1976) in Glutathione: Metabolism and Function

(Arias, I. M., and Jakoby, W. B., eds) pp. 77-114, Raven Press,

73. Gillham, B. (1971) B&hem. J. 121, 667-672

New York

74. Hyde, C. W., and Young, L. (1968) Biochem. J. 107, 519-522 75. Gillham, B., Clapp, J. J., Morrison, A. R., and Young, L. (1970)

Biochem. J. 118, 24p

78. Jerina, D. M., and Bend, J. R. (1977) in Biological Reactive Intermediates (Jollow, D. J., Kocsis, J. J., Snyder, R., and Vainio, H., eds) pp. 207-236, Plenum Press, New York

79. Wu, S.-C. G., and Straub, K. D. (1976) J. Biol. Chem. 251,6529- 6536

80. Sekura, R. D., and Jakoby, W. B. (1981) Arch. Biochem. Biophys. 211,352-359

81. Ketterer, B., Coles, B., and Meyer, D. J, (1983) Environ. HeaLth Perspect. 49,59-69

82. Meyer, D, J., Beale, D., Tan, K. H., Coles, B., and Ketterer, B. (1985) FEBS L&t. 184, 139-143

by guest on July 27, 2015http://w

ww

.jbc.org/D

ownloaded from

Page 8: A New Class of Rat Glutathione S-transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols

Glutathione S-transferase Yrs- Yrs

Amino acid analysis was performed 1" duplicate in JEOL model JLC-300 amino acid analyzer after hydrolysis of 100 vg of CST Yrs-Yrs 1" 3N mercaptoethanesulfonic acid at 1lO'C for 18 h. Data are arithmetic mean values of two experiments. The Amino acid composition was based on the assumption of the molecular weight of subunit Yrs as 26,000.

Amino acid content

[mol of amino acid/ mol of protein]

ASX Z(5) Thr 6 SW 17 GlX 3S(cJJ Pro 9 GlY 25 Ala 25 CYS l(h) v.91 8 Met 5 Ile 3 LeU 33 TY~ 4 Phe 11 LYS 12 His 11 Ax 12 TOP 1

2) Value reflects the sum of aspxtic acid plus aspar+ gine (or glutamic acid plus glutamine) because the amide was quantitatively converted to the correspond- ing acid during ac,d hydrolysis of the protein.

h) Determined as cysteic acid after performlc acid oxidation.

by guest on July 27, 2015http://w

ww

.jbc.org/D

ownloaded from

Page 9: A New Class of Rat Glutathione S-transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols

Glutathione S-transferase Yrs- Yrs 11981

0.3

0.2

0.1

0

66,000 *

45,000*

36,000 +

29,000 + - -lvc 24,000* - - - - ml 8”d “ix?

- -“a

by guest on July 27, 2015http://w

ww

.jbc.org/D

ownloaded from

Page 10: A New Class of Rat Glutathione S-transferase Yrs-Yrs Inactivating Reactive Sulfate Esters as Metabolites of Carcinogenic Arylmethanols

Hatayama, S Tsuchida and T IshikawaOkuda, K Ogura, T Watabe, K Satoh, I A Hiratsuka, N Sebata, K Kawashima, H  arylmethanols.as metabolites of carcinogenicYrs-Yrs inactivating reactive sulfate esters A new class of rat glutathione S-transferase:

1990, 265:11973-11981.J. Biol. Chem. 

  http://www.jbc.org/content/265/20/11973Access the most updated version of this article at

  .JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/20/11973.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on July 27, 2015http://w

ww

.jbc.org/D

ownloaded from