A number of potential substrates for the microsomal glutathione transferase have been investigated. Out of. 11 epoxides tested, only two, i.e. androstenoxide and.
THEJOURNAL OF BIOLOGICAL
Vol . 263, No. 14, Issue of May 15, pp. 6671-6675, 1988 Printed in U.S.A.
CHEMISTRY
0 1988 by The American Society for Biochemistry and Molecular Biology, h e .
Studies on the Activity and Activation of Rat Liver Microsomal Glutathione Transferase, in Particular witha Substrate Analogue Series* (Received for publication, October 26, 1987)
Ralf MorgensternS, Gerd Lundqvist,Victorine Hancock, and JosephW.DePierre From the Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden
A number of potential substrates for the microsomal glutathione transferase havebeen investigated. Out of 11 epoxides tested, only two, i.e. androstenoxide and benzo(a)pyrene-4,5-oxide,were found to be substrates. Upon treatment of the enzyme with N-ethylmaleimide, its activity towardonly certain substrates increased. is It appeared upon inspection of the bimolecular rate constants from the correspondingnonenzymatic reactions that the substrates for which the activity is increased are the more reactive ones. This hypothesis wasinvestigated further using a series of para-substituted 1-chloro-%nitrobenzenederivatives as substrates. Activation was seen only with the more reactive nitro-,aldehyde-, and acetaldehydesubstituted compounds and not with the amide and chloroanalogues, thus demonstrating the predictedeffect with a related seriesof compounds. Interestingly, k,, values are increased 7-20-fold by N-ethylmaleimide treatment, whereas the corresponding k,,JK, value is increased only for the p-nitro derivative.Effective molarity and rate enhancement values were found to increase with decreasing reactivity of the substrate, attaining maximal values of lo6 M and lo', respectively. It is concluded that theglutathione transferases are quite effective catalysts with their less reactive substrates. Hammett p values for the kc,, values of unactivated and activated enzyme were 0.49 and 2.0, respectively. The latter value is close to those found for cytosolic glutathionetransferases,indicatingthatactivation changes the catalyticmechanism so that itmore closely resembles that of the soluble enzymes. The p values for k J K , values were 3 and 3.5 for the unactivated and activated enzyme, respectively, values close to those observed for the nonenzymatic bimolecular rate constants andthereby demonstrating that these reactions have similar properties.The high coefficients of correlation between resonance u- values and all of these parameters demonstrate a strong dependence on substrate electrophilicity, as expected for nucleophilic aromatic substitution.
Glutathione transferases catalyze the conjugation of glutathione with a variety of molecules bearing different electrophilic centers. These second substrates are all hydrophobic
* These studieswere supported by grants from the Swedish Cancer Society and theSwedish Natural Science Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked "aduertisement" in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. $ To whom correspondence should be addressed.
(1).Thus, theglutathione transferases aid in the detoxication of numerous carcinogenic, toxic, and pharmacologicallyactive substances (1).A number of cytosolic enzymes have been isolated from mammalian and nonmammalian tissues, including plants, and this work has been extensively reviewed (2). There is also a membrane-bound microsomal glutathione transferase localized predominantly in the liver (of mammals) that shares many of the functional characteristics of its cytosolic counterparts (3). This microsomal glutathione transferase is present in relatively high amounts (3 and 5% of the total microsomal and mitochondrial outer membrane protein, respectively (4)) and has the distinguishing property of being activated by sulfhydryl reagents ( 5 ) . Upon examination of a range of substrates, it was found that treatmentof microsomal glutathione transferase with N ethylmaleimide increases its activities with certain substrates only, whereas its activities with other substrates remain unchanged. These puzzling observations were then fitted into a hypothesis proposing that activation is seen only with the more reactive substrates (e.g. those exhibiting a high nonenzymatic rate of reaction with glutathione) (preliminary observations reported in Ref. 6). To test this hypothesis further, we have examined here the effects of N-ethylmaleimide treatment of the enzyme on its catalytic properties with a series of para-substituted 1-chloro-2-nitrobenzenes with different reactivities, as well as with a range of new potential substrates. MATERIALSANDMETHODS
Microsomal glutathione transferase was purified from male Sprague-Dawley rat liver as previously described (7). Activation of the purified enzyme with 2 mM N-ethylmaleimide was performed a t 4 "cin 10 mM potassium phosphate, pH 8, 1%Triton X-100,O.l mM EDTA, 1 mM GSH, 20%glycerol, and 0.1 M KCI. When maximal activity was reached (within 5 min), the reaction was terminated by addition of GSH to give a final concentration of the free thiol of approximately 1 mM. Protein was determined by the method of Peterson (8)with bovine serum albumin as standard. Enzyme Assays-Activities with the fluoro-, bromo-, and iod0"2,4dinitrobenzenes were measured as described for the chloro derivative (9). Activities with para-substituted 1-chloro-2-nitrobenzenes were assayed in 0.1 M potassium phosphate, pH 6.5, containing 0.1%Triton X-100 and 5 mM GSH at the wavelengths described by Keen et al. (10). All known extinction coefficients for products were doublechecked by running reactions with limiting amounts of electrophilic substrate to completion and were in close agreement with published data (10).New extinction coefficients were determined according to Keen et al. (10). Activities with 4-chloro-3-nitrobenzophenone and 3chloro-4-nitrobenzanilidewere assayed at 381 nm (c = 3 In"'). 1Chloro-2-nitrobenzene (tZmnm= 4.4 mM") was found not to be a substrate. para-Nitrophenylacetatethiolysis was measured as in Ref. 11with 2 mM GSH, 0.2 mM substrate, and 0.1% Triton X-100 in the medium. Nonenzymatic reaction rates were calculated from previous experiments in the case of cumene hydroperoxide, methyl.iodide, p-nitrobenzylchloride, 1,2-dichloro-4-nitrobenzene, and trans-phenyl-3-b~-
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tene-2-one (7). The rates for androsteneoxide (1 mM), cholesterol TABLE I1 5,6a-epoxide (10 HM), estronepoxide (0.1 mM), and stearic acid 9,lOCompoundsfound not to be substrates for the microsomal epoxide (0.1 mM)were measured by the procedure reported for glutathione transferase hexachlorobutadiene in Ref. 7. AS-Androstene-3,17-dione isomerizaLower tion was measured as described (12), except that 0.1% Triton X-100 Compound limit of was included in the medium. The rate of the nonenzymatic reaction detection of benzo(a)pyrene-7,8-diol 9,lO-epoxide with glutathione was also prnollrnin mg determined using established procedures (13).
