Binding of acrylamide with glutathione-S-transferases

Chem.-Biol. Interactions, 32 (1980) 353--359

353

© Elsevier/North-HollandScientificPublishers Ltd.

BINDING OF ACRYLAMIDE WITH GLUTATHIONE-S-TRANSFERASES

RAKESH DIXIT, HASAN MUKHTAR, PRAHLAD K. SETH and C.R. KRISHNA MURTI Industrial Toxicology Research Centre, Post Box No. 80, Lucknow 226 001 (India)

(Received March 28th, 1980) (Revision received June 10th, 1980) (Accepted June 15th, 1980)

Introduction

GST, a family of cytosolic enzymes, plays an important role in cellular biotransformation and elimination of toxic electrophiles by glutathione conjugation and subsequent excretion as mereapturic acid [1,2]. GST bind a broad range of substrate and non~substrate ligands and subsequently function both as catalytic proteins for cellular biotransformation and as an organic anion or receptor protein [ 3--7 ]. ACR (CH2CHCONH2) is an extensively used monomer in polymer industry and is a potent neurotoxin [8]. Exposure of humans and animals to ACR leads to a typical peripheral neuropathy in nervous system [ 8]. To date very little is known about the biotransforrnation of ACR [9]. Our recent studies have demonstrated that ACR reacts with glutathione both nonenzymically and enzymically. Enzymically both liver and brain GST catalyze the formation of S-conjugates and therefore acrylamide acts as substrate for GST (R. Dixit et al., pets. comm.). In view of the wide range of ligands (both substrate and non~substrate) that bind to GST [3--7], it was of interest to study interaction of ACR with GST. The present report summarises the observations which suggest that the conjugating enzyme may be identical with the protein which binds ACR. Materials and Methods

Male albino rats of I.T.R.C. colony maintained on commercial pellet diet (Hindustan Lever Ltd., Bombay, India) were used in the present study. Rat liver and brain tissues were homogenized in 4 vols. of chilled 0.25 M sucrose and organelle flee cytosol fractions were recovered by conventional methods. Liver and brain cytosols were applied on top of columns of Sephadex Abbreviations: ACR, acrylamide; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2dichloro-4-nitrobenzene; GSH, glutathione; GST, glutathione-S-transferase;HPLC, high pressure liquid chromatography.

354 G-75 (36 × 2.5 cm) equilibrated with 10 mM Tris buffer (pH 8.2) which also served as the mobile phase. Fractions (10 ml and 5 ml) were collected for liver and brain cytosols respectively at a flow rate of 50 ml/h. Eluates were assayed for GST activity towards CDNB by the m e t h o d of Habig et al. [10]. GST activity with ACR as substrate was assayed b y measuring the disappearance o f glutathione as described by Boyland and Chasseud [1] for conjugation o f a ~ unsaturated substrates and modified as follows. Assay mixture in 3 ml contained 5 mM GSH, 5 mM ACR, 0.1 M phosphate buffer (pH 7.4) and suitable aliquots o f enzyme source. The non,enzymic rate was subtracted from the rate of enzymic activity which was assayed as above except that denatured enzyme was used. The reaction was stopped by addition o f 5% TCA. Residual glutathione was measured in supernatants by the m e t h o d of Ellman [11]. Specific activity was expressed a pmol GSH disappeared/min/mg protein. Binding studies. Fractions of GST recovered from gel filtration showing m a x i m u m enzyme activity were used to study binding of ACR to protein. ACR binding to GST was investigated by equilibrium dialysis of ACRprotein mixtures or quantifying the fluorescence o f enzyme caused by ACR. Dialysis experiment. ACR (140 ~mol) was incubated for 30 rain with peak GST fraction (15--20 mg protein) and was dialysed against distilled water at 0°C. At different intervals the concentration o f ACR was determined in the diffusate by high pressure liquid chromatography (HPLC). A waters associates HPLC equipped with a Bondapak C-18 analytical column (with a flow rate of 0.5 ml/min; 80% methanol (v/v) in water was used as solvent) attached with a UV detector set at 254 nm was used. Under these conditions single peak was obtained with retention time o f 5.7 min. Fluorescence quenching experiments. Quenching of the fluorescence of protein on addition of ACR was recorded in an Aminco Spectrofluorimeter (SPF 500) at wavelength maxima of 285 nm (excitation) and 330 nm (emission). Quenching due to bilirubin was studied as a control. ACR (2--20 mM) or bflirubin (0.2--1 mM) was added to a cuvette conraining adequate amounts of enzyme protein, 6% v/v glycerol, 0.2 M EDTA and 0.1 M phosphate buffer (pH 7.5) in a final volume adjusted to 3.0 ml. For non-enzymic protein-ligand interaction enzyme was denatured b y heating in a boiling water bath for 10 min. Binding studies with amino acids. ACR binding to aromatic amino acids was measured by determining the quenching of fluorescence of the amino acids caused by ACR. To small cuvettes containing 0.1 M phosphate buffer (pH 7.4) tyrosine (0.04--4 raM) or tryptophan (2--20 pM) ACR was added in a final volume of 3.0 ml. The decrease in fluorescence of tyrosine was measured at a wavelength maxima of 300 nm (emission) while that of t r y p t o p h a n was determined at 355 nm. A wavelength maxima of 285 nm (excitation) was used in both cases. Binding constants were calculated from the reciprocal plots as described by Ketley et al. [6].

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Results and Discussion Results depicted in Fig. 1 demonstrate that addition of ACR to enzyme protein resulted in the quenching o f fluorescence in a concentrationdependent (both protein and ligand) manner. B'flirubin, a known ligand o f GST also resulted in a concentration~lependent quenching of liver and brain enzyme protein (Fig. 2). Binding constants of ACR towards liver and brain GST calculated from reciprocal plots as described by Ketley et al. [6] were found to be 0,8 (raM) and 1.1 (raM) (Table I). The concentration of u n b o u n d ACR in the diffusate after dialysis for various time intervals is shown by Fig. 3. It should be noted that ACR has a strong affinity towards GST o f both liver and brain as 30--40% of total incubated ACR was found to bind to protein even after dialysis for 2 h.

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TABLE II A COMPARISON O F BINDING AND CONJUGATION CAPACITY O F LIVER AND BRAIN GST TOWARDS ACR Peak fraction is dermed as the cytosol fraction (obtained after Sephadex G-75 gel filtration chromatography) exhibiting highest activity with CDNB and DCNB as substrates. Data from a typical experiment. Parameter Liver GST activity o f peak fraction towards ACR a Brain GST activity o f peak fraction towards ACR Percent ACR bound to liver GST peak fraction at equilibrium Percent ACR bound to brain GST peak fraction at equilibrium a pmol glutathione disappeared/min/mg protein.

Activity 243 81 41.5 40.0

358 Data presented in Table II briefly summarize the relative conjugation and binding capacity o f liver and brain GST towards ACR. It is evident from the data that both liver and brain GST possess significant capacity for conjugation as well as binding of ACR. Liver GST was found to be 3 times more efficient in conjugating A C R as compared to brain GST b u t no striking difference in binding capacity of liver or brain GST was apparent. It was also noticed that the GST fraction which was maximally active in conjugation of CDNB, DCNB or ACR also had maximum capacity to bind ACR. Binding of ACR with GST was also studied in the presence and absence o f GSH. No significant binding of acrylamide with GST in the presence of GSH was noticed suggesting that binding of ACR with GST does n o t involve GSH. A C R was found to interact with tyrosine and tryptophan as evident from fluorescence quenching of both the amino acids dependent on both A C R and amino acid concentrations {Fig. 4). The possibility of a strong binding of ACR to GST cannot be ruled o u t as after 2 h of dialysis more than 30% of ACR was still b o u n d to the enzyme preparation. Certain alkylating agents and electrophilic substances, such as CDNB, interact at the catalytic site of the enzyme but also bind to GST at other sites and form stable covalent bonds [7]. The stable covalent binding of ACR to brain and liver GST is presumably of significance for the nonconjugative cellular disposal or intracellular transport of ACR. Binding of the neurotoxin to brain GST suggests that the enzyme may function as a receptor binding protein at the site of toxication. It appears that GST may have a bifunctional role in biotransformation of ACR, serving as a conjugating enzyme for catalytic disposal o f ACR and also serving as a binding protein for non-enzymatic elimination or intracellular transport of the neurotoxin. One of us (R.D.) is thankful to CSIR, New Delhi for award o f S.R.F. Thanks are also due to Mr. P.S. Shukla and Mr. Joseph George for typographical assistance. 1 E. Boyland and L.F. Chasseud, The role of glutathione and glutathione-Stransferases in mercapturic acid biosynthesis, Adv. Enzymol., 32 (1979) 173. 2 J.L. Wood, Biochemistry of mercapturic acid formation, in: W.H. Fishman (Ed.), Metabolic Conjugation and Metabolic Hydrolysis, Vol. 2, N e w York Academic Press Inc., 1970, pp. 261--291. 3 G. Litwack, B. Ketterer and I.M. Arias, Ligandin: A hepatic protein which binds steroids, bilirubin, carcinogens and number of exogenous organic anions, Nature, 234 (1971) 466. 4 Smith, G.J., Ohl, V.S. and G. Litwack, Ligandin: the glutathione-S-transferasesand chemically induced hepatocarcinogenesis, Cancer Res., 37 (1977) 8. 5 W.H. Habig, M.J. Pabst, G. Fleishner, Z. Gatmaitan, I.M. Arias and W.B. Jakoby, The identity of glutathione-S-transferases with ligandian, a major binding protein of rat liver,Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 158. 6 J.M. Ketley, W.H. Habig and W.B. Jakoby, Binding of nonsubstrate ligands to glutathione-S-transferases,J. Biol. Chem., 250 (1975) 8670. 7 W.B. Jakoby, The glutathione-S-transferases: A triple threat in detoxication, in:

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F.J. deSerres, J.R. Fouts, J.R. Bend, R.M. Philipot (Eds.), Elsevier/North-Holland Biomedical Press, In Vitro Metabolic Activation In Mutagenesis Testing, 1976. P.S. Spencer and H.H. Schaumburg, A review of acrylamide neurotoxicity. Part I. Properties, uses and human exposure, Can. J. Neurol. Sei., 1 (1974) 143. NIOSH criteria for a recommended occupational exposure to acrylamide US Department of Health, Education and Welfare, Oct., 1976. W.H. Habig, M.J. Pabst and W.B. Jakoby, Glutathione-S-transferases in mercapturic acid formation, J. Biol. Chem., 249 (1974) 7130. G.L. Ellman, Tissue Sulphhydryl groups, Arch. Biochem. Biophys., 82 (1959) 70.