pylthiouracil on the kinetics of the enzymatically cat- alyzed reaction between GSH and l-chloro-2,4-dinitro- benzene and found that propylthiouracil inhibited the.
Propylthiouracil A SUBSTRATE FOR THE GLUTATHIONE S-TRANSFERASES THAT COMPETES WITHGLUTATHIONE; (Received for publication, March 19,
1979, and in revised form, November 9,
1979)
Tadataka Yamada$ and Neil Kaplowitz From the Gastroenterology a n d Research Services, Veterans Administration Wadsworth Hospital Centera n d the UCLA School of Medicine, Los Angeles, California 90073
In previous studies, we have observed that propyl- in vitro the GSH conjugation of a number of lipophilic electhiouracil (PTU)binds to Sephadex 6-100 eluates of rat trophilic drugs and carcinogens (8-11). The enzymes in rat liver cytosol associated with glutathione (GSH) S- liver are thought to have a molecular weight of 45,000 and to transferase activity (EC 2.5.1.18). In order to study consist of two subunits, one of which binds GSH as a substrate, further this association between propylthiouracil and and the other which binds lipophilic co-substrates or nonthe GSH S-transferases, we examined the effect of pro- substrate ligands (3, 12). The latterportion of the enzymes is pylthiouracil on the kinetics of the enzymatically cat- thought to be variable and thus account for the differing, alyzed reaction between GSH and l-chloro-2,4-dinitro- albeit broad and overlapping, substrate specificities of each of benzene and found that propylthiouracil inhibited the the various transferases, while the former portion, which binds reaction uncompetitively with respect to the latter reactant, but competitively with respect to the former GSH, is thought to be constant. Whereas a number of sub(Ki= 0.62 mM). In addition to its activityas an inhibitor strates or non-substrate ligands have been shown to inhibit of the GSH S-transferases, propylthiouracil appeared the transferases by competing for the lipophilic binding site, to be a substrate. Reaction products between propyl- no substances other than GSH analogues or conjugates have thiouracil and several of the lipophilic substrates for been found to compete for the GSH binding site (13).In our the transferases were identified and the enzymatically studies, we have found that PTU inhibits the transferases catalyzed formation of these products obeyed Michae- with respect to GSH by binding to theGSH binding site asa lis-Menten kinetics. The specific activities of the par- substrate for reactions catalyzed by the enzymes. This obsertially purified GSH S-transferases AA, A, B, and C in vation may have important implications in the hepatic detoxcatalyzing the reaction between propylthiouracil and ification of potentially harmful xenobiotic agents. l-chlor0-2,4-dinitrobenzenewere 40, 52,141,and 58 EXPERIMENTALPROCEDURES nmol/mg/min, respectively, approximately 1 to 20%of Chemicals-Ethacrynic acid and Ipheno~yacetic-2-'~C] ethacrynic the specific activities of these enzymes in catalyzing the reaction between GSH and l-chloro-2,4-dinitroben- acid, 16 pCi/rng) were gifts of Merck, Sharpe and Dohme (Rahway, zene. Similar results were obtained with GSH S-trans- N.J.). Sulfobromophthalein, dithiothreitol, GSH, glutathione reducferases AA, B, and C purified to homogeneity. These tase, and NADPHwere obtained from Sigma ChemicalCo. (St.Louis, and p-nitrobenzylchloride, 1,2-dichloro-4 nitrobenzene, 1studies suggest that propylthiouracil inhibits the GSH Mo.) chloro-2,4-dinitrobenzene,and 1,2-epoxy-3-(p-nitrophenoxy) propane S-transferases in a unique fashion by competing with were obtained from Aldrich Chemical Co. (Milwaukee, Wisc.). PTU GSH as a substrate in reactions catalyzed by the en- andI-methyl-2-mercaptoiidazole(methimazole) were gifts of Eli zymes. Ldly and Co. (Indianapolis, Ind.). [I4C]Methyl iodide (49mCi/mmol)
6-n-Propyl-%thiouracil (PTU), a drug initially found to inhibit the organification of iodine in the thyroid gland ( l ) , has been shown to inhibit hepatic thyroxine 5'-monodeiodinase activity (2). In studies undertaken to characterize the mechanism of this latter action of PTU,' we found that it binds to a number of proteins in rat liver cytosol, one of them being the Sephadex G-100 protein peak associated with the glutathione (GSH) S-transferases (EC 2.5.1.18). We have examined this association more closely in our present study. The GSH S-transferases are a family of cytosolic enzymes found in liver (3) as well as othertissues (4-7) which catalyze * This work was supported by United States Public Health Service Grant GM/CA 24987 and VeteransAdministration Medical Research Funds. A preliminary communication of this work has appeared in abstract form (T. Yamada and N. Kaplowitz (1978)Gastroenterology 75,996).The costsof publication of this articlewere defrayed in part by the paymentof page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Postdoctoral fellow supported by National Institutes of Health, United States Public Health Service Training Grant AM 07180. ' T h e abbreviation used is: PTU, 6-n-propyl-2-thiouracil.
and kgly~ine-2-~H]GSH(944 mCi/mmol) were obtained from New England Nuclear (Boston, Mass.) and [S5S]PTU (109 pCi/mmol) was obtained from Amersham Corp. (Arlington Heights, Ill.). All other chemicals used in this study were readily available commercial products. No detectable impurities were observed with PTU and
