Chem.-Biol. Intemctions, 0 Elsevier/North-Holland

24 (1979) 317-327 Scientific Publishers

317

Ltd.

THE INTERACTIONS OF TRIETHYLTIN WITH RAT GLUTATHIONE-S-TRANSFERASES A, B AND C. ENZYME-INHIBITION AND EQUILIBRIUM-DIALYSIS

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EDWARD TIPPING, BRIAN KETTERER, LUCIA CHRISTODOULID ES, BARRY ELLIOTT a*, W. NORMAN ALDRIDGE ’ and JAMES W. BRIDGES b

M.

Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, London WlP 7PN, a Molecular Toxicology Section, Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey, SM5 4EF and b Department of Biochemistry, University of Surrey, Guildford, Surrey, GUZ 5XH. (United Kingdom) (Received June 3rd, 1978) (Accepted September 15th. 1978)

SUMMARY

Purified glutathione(GSH)S-transferases A, B and C from rat liver are inhibited by triethyltin (SnEtj). With 1-chloro-2,4dinitro benzene (CDNB) as the limiting substrate the inhibition is competitive in each case. At a GSH concentration of 5 . 10-j M the inhibition constants for transferases A and C at 25°C are similar and very low, 3.2 . loss M and 5.6 - 1O’-8M respectively, whereas for transferase B the inhibition constant is 3.5 :LO-5M. Equilibrium-dialysis experiments carried out at 4°C in the absence of GSH give apparent dissociation constants of 7.1 - 10e4 M and 3.4 - 10V4 M for transferases A and B respectively, but if 5 . 10e3 M glutathione is included in the dialysis solutions these values fall to 2.0 - lo-’ M ‘and 2.6 -lo-'M, which are within an order of magnitude of the kinetic Ki-values. Chromatographic experiments with Sephadex G-10 show that GSH and SnEt3 interact in aqueous solution under the conditions of the enzyme-kinetic and equilibrium-dialysis experiments. It is suggested that the inhibited enzymes are in the form of ternary complexes, enzyme-GSHSnEt3, in which GSH and SnEtj may or may not interact directly; or are possibly quatemary complexes, enzyme-(GSH)z-SnEt+ SnEtJ could be valuable as a selective inhibitor of transferases A and C in mixtures of the three transferases. ___----_-- _c__--__ l

* Present address: I.C.I. Central Toxicological Laboratory, Alderley Park, Mscclesfield, Cheshire SK10 4TJ, United Kingdom. Abbreviations: CDNB, lchloro-2,4dinitrohenzene; DCNB, l,.Zdichloro-4-nitrobenzene; GSH, glutathione; SnEts, triethyltin.

318

INTRODUCTION The GSH~-~~~e~s (EC 2.6.1.18) are a family of enzymes which catalyse the conjugation of a large number of electrophiles with WI-t They are distinguished from one another by their substrate specificities which nevertheless overlap [ 11. GSHS-transferase B (ligandin) is able to bind many compounds which are not substrates, mainly via the hydrophobic effect [251. B~d~~ofnon~ubs~~s totransferases AA,A ~dcgene~y~pe~ to be weakerthanto transferase B f5], while binding to ~sfe~s D, E and M [6] has not been studied. Henry and Byington [7] have recently shown that compounds having 3 CGe, 3 C-Sn or 3 C-Pb bonds are effective inhibitors of the 1,2dichloro4ni~obenzene(D~~NB)~SH-t~sfemse activity of rat liver 100 000 g supernatant, and that with DCNB as the liming substrate the ~hibition is competitive, Since DCNB is a good substrate only for transferases A and C 11), the inhibition patterns observed are virtually exclusive to these two transferases. The results of Henry and Byington are particularly interesting in that the competitive inhibition constants are 10’7-10’6 M, suggesting an affinity greater than might be expected from solely hydrophobic infractions involving the hydrocarbon parts of the o~norn~t~s. In the light of these observations, parallel kinetic and equilibrium binding studies of the interactions of arganometallic compounds with purified GSHS-transferases might provide information on the nature of the catalytic and binding sites of the proteins. Moreover, po~ibi~iti~s for the molecular mechanism of the toxic action of o~nome~li~ compounds might su st themselves if the interactions leading to the inhibition of the transferases were understood. Accordingly we have made kinetic measurements of the inhibie tion by SnEts of the enzymic activities of GSHYS-transferases A, B and C, and equilibrium-dialysis measurements of the binding of [““Sn]EtJ by transferases A and B. Another interesting observation made by Henry and Byington [7] was that high concentrations of sodium sulphide or 2,3-dimercaptopropanol were able to protect the DCNB-GSH-transferase activity of rat liver 100 000 g supematant from inhibition by triphenyltin. They suggested that this was because coordination of sulphur to tin occurred, giving rise to complexes ineffective as inhibitors. Although it has previously been reported that there is no interaction between SnEtJ and GSH [8 1, these effects of sulphur compounds, together with the high concentrations of GSH used in the measurement of enzyme activity, prompted us to reexamine the possibility of complex formation between SnEt, and GSH.

MATERIALSANDMETHODS Mu teriuls Triethyltin sulphate was prepared from triethyItin hydroxide, supplied by the Tin Research Institu~, Creenford, Midd:esex, U.K., as describe by

319 Aldridge and Cremer [ 81. Triethyl [ ‘13Sn]tin chloride (1.2 Ci/mol) was purchased from The Radiochemical Centre, Amersham, Bucks, U.K. Reduced GSH was from the Sigma Chemical Co. Ltd., Poole, Dorset, U.K. CPNB, DCNB and tins-4-phenyl-3-buten-2-one were from the Aldrich Chemical Co. Ltd., Don&, U.K. Sephadex G-10 and G-100 were from Pharmacia (G.B.) Ltd., London W5 5SS, U.K. DEAE cellulose (DE 23) was from Whatman Ltd., Maidstone, Kent, U.K. Polyethylene glycol (Carbowax 20M) was from G.T. Gurr, High Wycombe, Bucks, U.K. Ampholines were from LKB Instruments, Croydon, Surrey, U.K. Repamtion of enzymes

GSHS-transferase B (ligandin) was prepared as described previously [ 41. GSHS-transferase A was prepared as described by Ketterer et al. [ 91, who referred to the prepar&ion as “GSHS-aryl transferase”, the name given to the enzyme activity conjugating DCNB with GSH [lo]. From the work of Habig et al. [l] it is now clear that transferase A is only one of two distinct enzymes comprising “GSHS-aryl transferase”. The other is transferase C which we obtain as a by-product of the preparations of transferases A and B as follows. The first chromatographic step of the procedure involves passage of dialysed 100 000 g supernatant of rat liver through a DEAE cellulose column equilibrated with 1 mM-triethanolamine/l.25 mM-CaCl,/HCl buffer, pH 8.0 [4]. Transferases A and B pass through this column while more acidic proteins including transferase C are adsorbed. Transferase C was eluted with 200 cm3 of the original buffer made 0.05 M with respect to NaCl. This fraction was concentrated to approx. 5 cm3 by dialysis against solid polyethylene glycol and applied to a column (2.6 X 90 cm) of Sephadex G-100 equilibrated with 0.1 M-KC1/0.05 M-potassium phosphate buffer, yH 7.0. The fractions with CDNB-GSHS-transfer activity [l] corresponding to a molecular weight of about 46 000 g - mol” (VJV, 1.5-1.6) were pooled, dialysed against distilled water and subjected to isoelectric focusing on an LKB isoelectric focusing column containing a O-40% (w/v) discontinuous sucrose density gradient and Ampholines (1% w/v) in the pH-range 5-8. The fraction with transferase activity which focused at pH.7.*7.7 was collected, concentrated by ultrafiltration to 1 cm3 and applied to the Sephadex G-100 column once more to remove Ampholines and sucrose. The single protein peak was the final transferase C preparation. Transferases A, B and C were checked for purity by SDS-urea-polyacrylamide gel electrophoresis according to Maize1 [ 111. Transferases A and C cross-reacted with an antibody raised against transferase A, as reported by Habig et al. [ 11, but were distinguishable by (a) their isoelectric points of 8.3 and 7.6 respectively and (b) the ten-fold higher specific activity of transferase C, compared to transferase A, with trans-4-phenyl-3-buten-2-one as substrate (cf. ref. 1). The absence of significant amounts of either transferase D or E in preparations of transferase C was shown by the inability of these preparations to catalyse the conjugation of p-nitrophenethyl bromide with GSH (cf. ref. 1).

Kinetic measurements

These were carried out as described by Habig et al. [l] at 2S”C except that 0.1 M,KC1/0.025 M-potassium phosphate buffer, pH 6.5 was used instead of 0.1 M-potassium phosphate, pH 6.5. Equilibrium

dialysis

Experiments were carried out as described previously [4] at 4’C except that 0.1 M-KC1/0.025 M-potassium phosphate buffer, pH 6.5 was used, as in the kinetic experiments. [ * ‘%n]Etj concentrations were determined as described by Rose and Aldridge [12] except that a Beckman Biogamma Counter was used. Control experiments showed that equilibrium was reached within 24 hours, whether or not GSH was included in the buffer, and that adsorption of SnEtS to the dialysis cells or membranes was negligible. Sephadex G-1 0 chromatography

A column (0.9 X 30 cm) was packed with Sephadex G-10 and equilibrated with 0.1 M-KC1/0.025 M-potassium phosphate buffer pH 6.5 or with the same buffer containing 5 * 10 -3 M glutathione. When glutathione was present the buffer reservoir was flushed with Nz throughout the experiment. Radioactive SnEts (0.5 cm3, 0.5 I.cmol) was applied to the column, 2 cm3 fractions were collected and their radioactivities measured. RESULTS AND DISCUSSION

At a saturating concentration (5 - loo3 M) of GSH, and with CDNB as the limiting substrate, SnEtJ is a competitive inhibitor of transferases A, B and C (Figs. l-3). Transferases A and C resemble each other in sensitivity towards inhibition by SnEtj, both having inhibition constants some three orders of magnitude smaller than that of transferase B (Table I) *. For transferases A and C our results are in broad agreement with those of Henry and Byington [7] who, by using DCNB as substrate with whole cytosol as the enzyme source, assayed what was essentially a mixture of the two enzymes (see Introduction). From measurements by equilibrium-dialysis we find that in the absence of GSH the interactions of SnEt, with transferases A and B are quite weak, dissociation constants of approx. 10e3 M and 3 - 10s4 M respectively being * It should be noted that for transferases A and C, the total concentrations of inhibitor (BnEtj) approach that of the enzyme (see the legends to Figs. 1 and 3). This means that the usual assumption that the total inhibitor concentration is equal to the concentration not bound to protein is not strictly valid. The effect is most serious for transferase C, where at a lotal inhibitor concentration of 1.25 - lo-’ M the actual unbound concentration could !re as low as 1.04 - IO- M at low concentrations of CDNB; this is calculated for an inhilrition constant, assumed to be a dissociation constant, of 5.6 . lo-’ M (cf. Table I), at a protein concentration of 3 0 lo-’ M, in the absence of CDNB. This rather small variation (~17%) in unbound concentration should not have any significant effects on the values obtained for the inhibition constants.

321

V

Fig. 1. Inhibition by SnEt, of GSH-S-transferase A. The velocity, v, has units of moi min- ’ - mol enzyme-‘, The concentration of CDNB is in mol * 1-l (M). The enzyme concentration was 6 * lo’* M; theconcentrationsof SnEt, were zero (0). 6.3 - lo-” M (B) and 1.9 - lo-’ M (A); the concentration of GSH was 5 . 10m3 M. Each point in Figs. l-3 is the mean of three determinations. The insets of Figs. l-3 are plots of the reciprocals of the slopes of the Eadie-Ilofstee plots (i.e. the apparent K, values) against the SnEts concentrations: the slopes of these secondary plots are given by K,,,/Ki [ 131.

obtained (Fig. 4). When 5 * 10-j M GSH is introduced into the equilibriumdialysis solutions the dissociation constants for the two transferases (2.0 lo-’ M for A and 2.6 10e5 M for B, both at 4°C) become comparable to the kinetically determined inhibition constants (cf. Table I). In the presence of GSH we can definitely conclude that both transferases bind SnEt, at a single site (Fig. 4). In its absence the evidence is less convincing since the amounts of the proteins available were limited and only low values of P could be obtained. It is hard to see how the binding of GSH by the enzymes could decrease the number of SnEt3 binding sites and so in Fig. 4 the Scatchard plots in the absence of GSH are drawn for one site. Fig. 5 shows the effect of GSH on the chromatography of SnEtJ on Sephadex G-10. The decreased elution volume of the organometal when the eluting buffer contains 5 - 10m3 M GSH suggests that under these conditions SnEt, and GSH are able to form a complex. At a GSH concentration of 5 * 10Ds M complexation is not apparent [S ] and so the interaction is probably quite weak. In view of the evidence presented by Henry and Byington [7] for the interactions of organotin with sulphido and 2,Sdimercaptopropanol, l

322

Fig. 2. Inhibition by SnEt3 of GSHS-transferase B. The enzyme concentration was 4.5 lo-’ M; the concentrations of SnEt3 were zero (@), 3.8 - 10s5 M (m) and 9.4 - 10e5 M (A); the concentration of GSH was 5 - 10-j M.

and of that given by Elliott [ 151 for the involvement of cyst&y1 residues in the binding of SnEt3 by cat haemoglobin, it may be that the SnEt,-GSH complex involves a tin-sulphur bond. Mechanism of binding of triethyltin In the ab;?ence of GSH it is probably the three ethyl groups of SnEt3 which cause it to bind to transferases A and B, both of which are known to have a hydrophobic binding site [Z-5]. From the kinetic experiments which show that &Et3 is a competitive inhibitor of transferases A, B and C when CDNB is the limiting substrate we can conclude that SnEt3 and CDNB have a common hydrophobic binding site (the first catalytic site) on each transferase. Therefore when SnEt, is bound to the proteins in the presence of excess GSH it is adjacent to GSH bound at the second catalytic site. Soluklons of SnEt, in chioride/phosphate buffer probably contain complexes of SnEt, with chloride, phosphate and hydroxide, as well as hydrated SnEt3 i’tself [ 16,171 to which must be added the SnEt3-GSH complex when a GSH is present. The binding of SnEt3 or any of its complexes to protein is therefore accompanied by changes in a number of linked equilibria, and so the dissociation and inhibition constants we have measured refer to a com-

323

V

Fig. 3. Xnhibitio~ by S&t3 of GS~~-transferase C. The enzyme concentration was 3 10e8 M; the con~ntrations of SnEt3 were zero (01, 1.25 - lo-’ M (8) and 3.75 . lo-‘M (A); the concentration of GSH was 5 - 10s3 M.

bination of undefined processes. This observed binding affinities (decreases brought about by GSH can only be exact interpretation awaits data for the

means that the enhancements in the in apparent dissociation complexes) discussed in qu~itative terms. More solution equilibria of SnEt3.

TABLE I KINETIC PARAMETERS AND INHIBITION CONSTANTS FOR GSH-S-TRANSFERASES A, B AND C. THE LAMING SUESTRATE WAS CDNB, THE CONCE~RATION OF GSH WAS 5 - lO-3 M. Data are derived from the plots shown in Figs. l-3. The figures in brackets are the results of Fabig et al., [l]. transferaw A simm0l.l-l) LIZ?. min’” & (mol - 1-l )

1.9 * lo-$ (6.0 1O-5) 1120 (3000) 3.2 = lo-’ l

- mol enzyme-’

)

transferase B 6.0 - 1O-4 (8.0 10-e) 980 (860) 3.5 lo-$ l

l

transferase C 3.7 * 1o-5 (1.0 10-e) 320 (500) 5.6 10-O l

l

GSH-S- transfemsc? B

Fig. 4. Scatchard plots [14] for the binding of SnEt3 by GSHS-transferases A and B in the presence and absence of GSH. -, [GSH] = 5 [GSH] = zero; -, 10e3 M. Proteir conaentrations were lo- 5-2.5 . 10M5M. Note the different ordinates in the upper panei, B = mot 113Sn bound per mole of proteid. From the siopes of the plots the apparent ~~o~~;tion constants are, for trakferase A, 7.1 - lo4 M (no GSH) and 2.0 - low7 M (5 * 10W3M GSH) and for transferase B, 3.4 * IO4 M (no GSH) and 2.6 e lo+ M (5 1O-3 M GSH). l

Perh,aps the simp’lest explanation of the decrease in dissociation constant is that the &at3 and GSH moieties of the S~t3~S~ complex {which we assume, for the sake of argument, to have 3. : I stoichiometry) occupy the first and second catalytic sites of the enzyme respectively, but are bonded to one another as in firee solution. This could come about either by the binding of free SnEt, to G4iH-bound enzyme, or by the SnEt3-GSH complex binding to the GSH-free enzyme; clearly the two pathways are ~e~od~~i~ly

325

O

10

20 Fraction

0

number

Fig. 5. Sephadex G-10 chromatography of SnEts in the absence of GSH (0) and in the presence of 5 - 10B3 M GSH (a). Pretreatment of the column with 5 - low3 M GSH followed by a brief elution with GSH-free buffer gave an elution profile identical to tbat obtained with a column which had not previously been exposed to GSH, which indicates that the effect on the triethyltin elution is not due to modification of the Sephadex. For details see Materials and Methods.

equivalent. If this mechanism is correct then the dissociation constant for GSH in the presence of SnEt, would appear different to that in its absence. This would not be expected in an alternative mechanism in which bound GSH is not directly involved in SnEt, binding but causes a conformational change in the hydrophobic site, thereby making it more attractive to SnEt, (unless the reverse also occurs, i.e. SnEtB binding to the hydrophobic site alters the conformation of the GSH binding site). It is conceivable that such a conformational change could allow the Sn atom to bond to two ammo acid residues in the protein itself and thus achieve its favoured pentacoordinate state; such bonding is believed to occur with histidine residues in a number of proteins [16,18,19] and with cysteine in cat haemoglobin [15]. Since the spatial restrictions on such bonding are rather strict only a minor change in the relative positions of residues in or near the protein binding site might be required. A mixture of these two mechanisms can also be envisaged in which the Sn atom achieves pentacoordination by bonding to one residue in the protein and to protein-bound GSH. A third possibility invohes the formation of a protein-SnE&-(GSH), complex wherein the SnE&GSH complex in solution binds to GSH-bound

enzyme, forming a second Sn-S bond to achieve pcntacoordination. In this case not only would GSH appear to have a different dissociation constant in the presence of SnEtj but also would have two apparently equivalent sites. From the above it is clear that further experiments are required to distinguish the various possibilities for the binding mechanism. Transferases A and B are affected differently by GSH with regard to their binding of SnEtj in that the apparent dissociation constant is decreased by three orders of magnitude for the former but by only one order of magnitude for the latter, and so may bind via different mechanisms. On the other hand the interactions of SnEts with transferase C may be like those with transferase A, in view of the similarity between the kinetic properties of the two enzymes [l] and of the comparable inhibition constants. More work must also be done on in particular to determine its stability and the SnEt&XH complex, stoichiometry. Use of SnEt, as a selective inhibitor On a practical level our results show that SnEt3 could be a valuable tool as a selective inhibitor in experiments on mixtures of transferases A, B and C, e.g. in homogenates or soluble supernatants. This is because in the presence of sufficient GSH, transferases A and C can be inhibited almost completely by concentrations of SnEtj which would leave transferase B virtually unaffected. On the basis of the inhibition constants shown in Table I, it can be calculated that at an unbound SnEt3 concentration of low6 M, transferase A would be 97% inhibited, transferase C 95% inhibited, but transferase B only 3% inhibited. ACKNOWLEDGEMENTS

B. Ketterer, E. Tipping and L. Christodoulides thank the Cancer Research Campaign (of which B. Ketterer is a Life Fellow) for a generous grant. B.M. Elliott thanks the Science Research Council for a research grant. REFERENCES 1 W.H. Hzbig, M.J. Pabst and W.B. Jakoby, Glutathione-S-transferases. The first enzymatic step in mercapturic acid formation . 2. Biol. Chem. 249 (1974) 7130. 2 G. Litwack, B. Ketterer and I.M. Arias, Ligandin: A hepatic protein which binds steroids, oilirubin, carcinogens and a number of exogenous organic anions. Nature (London) 234 (1971) 466. 3 K. Kamisuka, I. Listowsky, Z. Gatmaitan and I.M. Arias, Interactions of bilirubin and other ligands with ligandin. Biochemistry 14 (1975) 2175. 4 E. Tipping, Il. gtetterer, L. Christodoulides and G. Enderby, The non-covalent binding of small molecules by ligandin. Interactions with steroids and their conjugates, fatty acids, bro nosulphophthalein, carcinogens, glutathione and related compounds. Eur. J. Biochem. 67 (1976) 583. 5 J.N. Ketley, W.H. Habig and W.B. Jakoby, Binding of nonsubstrate ligands to the glutathione transferases. J. Biol. Chem. 250 (1975) 8670. 6 B. Gillham, The mechanism of the reaction between glutathione and 1-menaphthyl

327 sulphate catalysed by a g1utathione-Stransferase. Biochem. J. 135 (1973) 797. 7 R.A. Henry and K.H. Byington, Inhibition of glutathione-S-aryl transferase from rat liver by organogermanium, lead and tin compounds. Biochem. Pharmacol. 25 (1973) 2291. 8 W.N. Aldridge and J.E. Cremer, The biochemistry of organotin compounds. Diethyltin dichloride and triethyltin sulphate. Biochem. 9.61 (1955) 406. 9 B. Ketterer, L. Christodoulides, G. Enderby and E. Tipping, Mercapturic acid biosynthesis: the separate identities of glutathione-S-aryl chloride transferase and ligandin, Biochem. Biophys. Res. Commun., 57 (1974) 142. 10 E. Boyland and L.F. Chasseaud, The role of ghrtathione and glutathione-S-transferase in mercapturic acid biosynthesis, Adv. Enzymoi., 32 (1969) 173. 11 J.V. Maizel, Polyacrylamide gel electrophoresis of viral proteins, Methods Viral., 5 (1971) 180. 12 M.S. Rose and W.N. AIdridge, The interaction of triethyltin with components of animal t&sues, Biochem. J., 106 (1968) 821. 13 M. Dixon and E.C. Webb, Enzymes, 1964 p. 315. 14 G. Scatchard, The attraction of proteins for small molecules and ions, Ann. N.Y. Acad. Sci., 51 (1949) 660. 15 B.M. Elliott, Ph.D. Thesis, University of Surrey, 1977. 16 M.S. Rose, Evidence for histidine in the triethyltin-binding site of rat haemoglobin, Biochem. J., 111 (1969) 129. 17 W.N. AIdridge, B.W. Street and D.N. Skiileter, Oxidative phosphorylation. Halidedependent and halide-independent effects of triorganotin and triorganolead compounds on mitochondrial function, Biochem. J., 168 (1977) 353. 18 B.M. Elliott and W.N. Aidridge, Binding of triethyltin to cat haemoglobin and modification of the binding sites by diethyl pyrocarbonate, Biochem. J., 163 (1977) 583. 19 M.S. Rose and E.A. Lock, The interaction of triethyltin with a component of guineapig liver supernatant. Evidence for histidine in the binding sites, Biochem. J., 120 (1970) 151.

The interactions of triethyltin with rat glutathione-S-transferases A, B and C. Enzyme-inhibition and equilibrium-dialysis studies.

Chem.-Biol. Intemctions, 0 Elsevier/North-Holland 24 (1979) 317-327 Scientific Publishers 317 Ltd. THE INTERACTIONS OF TRIETHYLTIN WITH RAT GLUTAT...
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