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Biochem. J. (1992) 283, 217-222 (Printed in Great Britain)
Glutathione transferase isoenzymes from Bufo bufo embryos at early developmental stage
an
Carmine DI ILIO,*1I Antonio ACETO,* Tonino BUCCIARELLI,* Beatrice DRAGANI,* Stefania ANGELUCCI,* Michele MIRANDA,t Anna POMA,t Fernanda AMICARELLI,t Donatella BARRA$ and Giorgio FEDERICI§ *Istituto di Scienze Biochimiche Facolta' di Medicina, Universita 'G. D'Annunzio', Chieti, Italy,
tDipartimento di Biologia e Fisiologia Cellulare Universita dell'Aquila, L'Aquila, Italy,
:Dipartimento di Scienze Biochimiche e Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Universit'a di Roma 'La Sapienza', Roma, Italy, and §Dipartimento di Biologia, Universit'a di Roma 'Tor Vergata', Roma, Italy
Six forms of glutathione transferase (GST) were resolved from the cytosolic fraction of Bufo bufo embryos at developmental stage 4 by GSH-Sepharose affinity chromatography followed by f.p.l.c. chromatofocusing in the 9-6 pH range. They have apparent isoelectric points at pH 8.37 (GST I), 8.22 (GST II), 8.10 (GST III), 7.84 (GST IV), 7.37 (GST V) and 7.12 (GST VI), and each displayed an apparent subunit molecular mass of 23 kDa by SDS/PAGE. The Bufo bufo embryo enzymes showed very similar structural, catalytic and immunological properties, as indicated by their substratespecificities, inhibition characteristics, c.d. spectra, h.p.l.c. elution profiles and immunological reactivities, as well as by their N-terminal amino acid sequences. Although Bufo bufo embryo GSTs do not correspond to any other known GSTs, the results of our experiments indicate that amphibian GSTs could be included in the Pi family of GSTs. This conclusion is supported by the analysis of c.d. spectra, and by the fact that mammalian Pi class GSTs and amphibian GSTs showed about 80 0 identity in their N-terminal amino acid sequences. Furthermore, antisera prepared against Bufo bufo GST III cross-reacted in immunoblotting analysis with Pi class GSTs, and vice versa. INTRODUCTION The cytosolic glutathione transferases (GSTs; EC 2.5.1.18) constitute a family of multifunctional proteins that catalyse the conjugation of GSH to a large variety of hydrophobic electrophiles (Jakoby & Habig, 1980; Awasthi & Singh, 1985; Mannervik, 1985; Ketterer et al., 1988), including herbicides, insecticides, carcinogens and mutagens (Chasseaud, 1979; Hayes & Wolf, 1988; Lamoureux & Rusness, 1989). In mammalian species the cystolic GST isoenzymes can be grouped into three speciesindependent classes named Alpha, Mu and Pi (Mannervik et al., 1985). GSTs have also been purified from micro-organisms (Di Ilio et al., 1988a, 1989; Piccolomini et al., 1989), and the data available indicate that bacterial GSTs cannot be included into any of the three above-mentioned classes (Di Ilio et al., 1988a, 1989, 1991; Piccolomini et al., 1989). In a previous study we found relatively high levels of GST activity in the embryos of Bufo bufo in all stages of development (Del Boccio et al., 1987a). The maximum level of activity was found at stage 4, after which a gradual decrease of activity up to stage 25 occurred. The isoelectric-focusing analysis of cytosolic fractions indicated that the Bufo bufo embryos are dominated by GSTs with nearneutral/cationic behaviours (Del Boccio et al., 1987a). In addition, the isoelectric-focusing profiles of adult toad liver and kidney cytosolic fractions proved to be significantly different from those obtained from embryos (Del Boccio et al., 1987a). However, to obtain a better understanding of whether during embryonic development specific alteration in the isoenzyme composition occurs, it is essential to characterize the GST system of Bufo bufo embryos from the early stages of development. Furthermore, since Bufo bufo embryos are externally isolated until a late stage of development, the characterization of the GSTs of this organism appears to be particularly interesting in obtaining a better understanding of the role of these proteins in detoxication processes. Abbreviation used: GST, glutathione transferase. 1 To whom correspondence should be addressed.
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In the present study we have therefore purified the Bufo bufo embryo GSTs from stage 4 by using GSH-Sepharose affinity chromatography and chromatofocusing (f.p.l.c.) techniques. The enzymes were characterized with respect to subunit molecular mass, substrate-specificity, inhibition characteristics, immunological reactivity, c.d. spectra, h.p.l.c. profiles and N-terminal amino acid sequences.
MATERIALS AND METHODS Embryos Adult Bufo bufo were collected near L'Aquila (Italy). Ovulation and fertilization occurred in the laboratory. The eggs were kept at 12-14 °C in tap water. The embryonic jelly was removed by treatment with 40% (w/v) sodium thioglycollate at pH 8.6 in a ratio of 1: 1 (v/v) and washed three times with 10 mM-sodium phosphate buffer, pH 7.0, before the preparation of the cytosol. The embryos of Bufo bufo were assigned to stages by reference to Rugh (1961). GST purification Embryos at developmental stage 4 were suspended in 10 mmpotassium phosphate buffer, pH 7.0, supplemented with 1 mmdithiothreitol (buffer A) and homogenized in a Potter homogenizer by hand with ten pestle strokes. The extract was centrifuged at 105 000 g for 60 min at 4 °C, and the resulting supernatant was applied to a GSH-Sepharose affinity column (Simons & Vander Jagt, 1977) that had been pre-equilibrated with buffer A. The column was exhaustively washed with buffer A supplemented with 50 mM-KCI. The enzyme was eluted with Tris/HCl buffer, pH 9.6, containing 5 mM-GSH. The fractions containing GST activity were pooled, concentrated by ultrafiltration, dialysed against buffer A and subjected to a chromatofocusing run. Isoenzymes of GST were separated by h.p.l.c. with a Kontron h.p.l.c. system. The sample was filtered through a
C. Di Ilio and others
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0.2,um-pore-size Millipore filter before application on to a Mono P HR 5/20 column (Pharmacia). The column was preequilibrated with 25 mM-Tris/HCI buffer, pH 9.3. Elution was carried out with Polybuffer 96 diluted 1:12.5 adjusted to pH 6.0 with acetic acid. The flow rate was 1 ml/min. Each fraction was 0.5 ml. The peaks of activity thus separated were concentrated by ultrafiltration, dialysed against buffer A and used for further characterization. SDS/PAGE Subunit molecular masses of GSTs were determined by SDS/PAGE as described by Laemmli (1970). SDS concentration was 0.1 00, and the spacer gel and the separating gel were 3 %O and 12.5 0/0 respectively. Phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soya-bean trypsin inhibitor (20.1 kDa) and ac-lactalbumin (14.4 kDa) were used as standards for characterization of subunit molecular mass. Antisera Attempts to prepare antisera against the individual isoenzymes of Bufo bufo were successful only for GST III, and this antiserum was raised in rabbits via two injections of about 100 ,ug of protein in Freund's complete adjuvant. Antisera against members of human Alpha, Mu and Pi class GSTs, as well as antisera against bacterial GSTs, were available in our laboratory and were the same as those used in previous studies (Del Boccio et al., 1987b; Di Ilio et al., 1988a,b, 1991; Aceto et al., 1989). These antisera recognize GST isoenzymes belonging to the same class but do not recognize members of the other classes.
Immunoblotting analysis Proteins were electrophoretically transferred from polyacrylamide gels on to nitrocellulose membranes (Bio-Rad Transblot system) according to the method of Towbin et al. (1979). Electroblotting was done for 16 h at 30 V in 25 mM-Tris base/ 192 mM-glycine, pH 8.3, containing 20 % (v/v) methanol. All incubations were performed at 25 °C with intermediate rinses in 400 mM-NaCl/50 mM-Tris base buffer, pH 7.5 (buffer B), containing 0.05 0 Tween 20 (buffer C). Non-specific binding was blocked by placing nitrocellulose paper in buffer B, supplemented with 3 %0 (w/v) BSA. Nitrocellulose papers were incubated with appropriately diluted antiserum in buffer B containing 3 0 BSA at 25 °C overnight. The nitrocellulose paper was washed with buffer C and then incubated for 1 h at 25 °C, with gentle shaking, in the same buffer containing 1 %0 (w/v) gelatin and horseradishperoxidase-conjugated goat anti-(rabbit IgG) antibody (BioRad Laboratories) diluted 1:3000. After treatment with peroxidase-conjugated antibodies, the nitrocellulose paper was washed three times with buffer C (5 min each) and twice with buffer B, then immersed in development solution [100 ml of buffer B containing 60 mg of 4-chloro- 1-naphthol (Bio-Rad Laboratories) and 60 ml of 30 % (v/v) H202]. The blot was then washed once with distilled water, air-dried and photographed. Enzyme assay GST activity with 1-chloro-2,4-dinitrobenzene, ethacrynic acid, androst-5-ene-3, 1 7-dione and trans-4-phenylbut-3-en-2-one was measured as described by Habig & Jakoby (1981). GST activity with 4-nitroquinoline 1-oxide and with trans-non-2-enal as substrates was determined as described by Stanley & Benson (1988) and Brophy et al. (1989) respectively. GST activity with cumene hyroperoxide as substrate was measured as previously reported (Di Ilio et al., 1986a). Protein concentration was
determined by the method of Bradford (1976), with y-globulin as a standard. Analysis by h.p.l.c. The reverse-phase h.p.l.c. analysis of purified Bufo bufo GST was performed by using the method described by Ostlund Farrants et al. (1987). Samples of Bufo bufo GSTs (about 20 ,ug) were injected on to a Waters ,tBondapak C18 (0.39 cm x 30.0 cm) attached to a Kontron h.p.l.c. system that had previously been equilibrated with 8 °,' (v/v) acetonitrile in 0.1 % (v/v) trifluoroacetic acid. The column was developed at 1 ml/min by a 60 min gradient from 8 % to 55,00 (v/v) acetonitrile in 0.1 00 (v/v) trifluoroacetic acid; this was followed by a 55-70 O0 (v/v) acetonitrile gradient in 0.1 0 (v/v) trifluoroacetic acid formed over 5 min. The eluate was monitored at 220 nm.
C.d. spectra C.d. spectra were obtained with a Jasco J-500 A instrument equipped with Jasco DP-500 N data processor. The content of secondary structure was estimated from the spectra between 200 and 250 nm as described by Greenfield & Fasman (1969). N-Terminal amino acid sequencing N-Terminal amino acid sequencing was performed on an Applied Biosystems model 475A gas-phase protein sequencer equipped with an Applied Biosystems model 120A analyser for the on-line detection of amino acid phenylthiohydantoin derivatives. A sample (about 2 nmol) of native protein was loaded on to a trifluoroacetic acid-treated glass-fibre filter coated with Polybrene. RESULTS Purification Table 1 summarizes the results of a typical purification of GST present in the embryos of Bufo bufo cytosol monitored with 1chloro-2,4-dinitrobenzene as substrate. GST was purified about 12-fold with a total recovery of about 90 % after the affinitychromatography step. The GSTs purified by affinity chromatography were subjected to chromatofocusing at pH 9.0-6.0. At least six major peaks, I-VI, appeared, as shown in Fig. 1. The apparent pl values of peaks I, II, III, IV, V and VI were 8.37, 8.22, 8.10, 7.84, 7.37 and 7.12 respectively. About 15 0 of the protein was eluted with 1 M-NaCl and was not further investi-
gated. Table 1. Purification of GSTs from Bufo bufo embryos For experimental details see the text.
Specific activity
Step Cytosol Affinity chromatography Chromatofocusing GST I (pl 8.37) GST I1 (pl 8.22) GST III (p1 8.10) GST IV (p1 7.84) GST V (pl 7.37) GST VI (pl 7.12) NaCl
(umol/min per mg)
1.48 17.5
47 43 46 27 30 10 6.0
Total
activity
(pmol/min) 392
350 8.5 64.5 87.4 54 3 2 5.4
Total protein
Yield
(mg)
(°0)
265 20 0.18 1.5 1.9 2.0 0.1 0.2 0.9
100 90
64
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Glutathione transferase isoenzymes from Bufo bufo embryos
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9
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1IV
0.4
100 83 20
GST II 0.1 25 63.5 3 60 150 30 > 100 83 20
> 1000
> 1000
15
10
CUM) GST III 0.1 25 100 3 45 150 37.5 > 100 50 20 > 1000 14
GST IV 0.1 25 81.7 3 30 150 30 > 100 50 20 > 1000 15
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221
Glutathione transferase isoenzymes from Bufo bufo embryos
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The resolved GST forms from Bufo bufo embryos appear to be dimers with broadly similar structural features. For example, none of the enzymes isolated arose from binary combination of subunits with different molecular mass. All forms turned out to be dimers of 23 kDa. On SDS/PAGE they migrated slightly faster than Xrat .¢ X > GST I subunit. Bufo bufo GSTs appear to be similar in many respects. In addition to the electrophoretic mobility, this is indicated by similarities of substrate-specificities, inhibition in the presence of dithiothreitol. The apparent similarities between the four forms also suggest the possibility that they may > > arisen from post-translational modification of a single have precursor. Although the results of N-terminal amino acid sequences do not provide evidence of any difference among the v P Xfour forms, at present we cannot exclude that the differences may reside beyond this region, or that they express different alleles of a polymorphic protein; in fact, the enzyme is extracted from a population of hundreds of brother embryos whose parent genotypes as concerns GST locus(i) are not known, nor is it known if in Bufo bufo iso-loci for GST exist. The results of our experiments demonstrated that Bufo bufo embryo GSTs have greater structural and immunological similarity to the GSTs of the Pi class. For the first 36 amino acid o
4
222
residues Bufo bufo embryo GSTs have about 80 % identity with class Pi GSTs. It has to be noted that comparison of GST isoenzymes included in the same class between mammalian species has about 70-80% residue identity in their N-terminal region, whereas GSTs belonging to different classes have less than 3000 sequence identity (Mannervik & Danielson, 1988). It should be noted that at position 2 Bufo bufo GSTs contain a residue of glutamic acid, whereas all other mammalian Pi class GSTs so far isolated were found to contain proline (Mannervik & Danielson, 1988). That Bufo bufo GSTs may be included in the GST Pi family is also supported by the results of c.d. spectra and especially by immunological studies. Our results demonstrate a clear immunological cross-reactivity between mammalian Pi class GSTs and the GSTs of Bufo bufo. In fact, Bufo bufo GSTs can be immunoprecipitated by anti-(GST T) sera. Conversely, human GST n7, rat GST 7-7 and mouse GST Mll, all belonging to the Pi family, are immunoprecipitated by Bufo bufo GST III antisera, so that it seems plausible to suggest that amphibian and mammalian Pi class GSTs share a common ancestor gene. This work was supported by C. N. R. Progetto Finalizzato 'Prevenzione e Controllo dei Fattori di Malattia' (FATMA), Sottoprogetto 'Qualitai' dell'Ambiente e Salute Contratto no. 91.00146.PF41.
REFERENCES Aceto, A., Di Ilio, C., Angelucci, S., Tenaglia, R., Zezza, A., Caccuri, A. M. & Federici, G. (1989) Clin. Chim. Acta 183, 83-86 Aceto, A., Di Ilio, C., Lo Bello, M., Bucciarelli, T., Angelucci, S. & Federici, G. (1990) Carcinogenesis 11, 2267-2269 Awasthi, Y. C. & Singh, S. V. (1985) Comp. Biochem. Physiol. B 82, 17-23 Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 Brophy, P. M., Southhan, C. & Barret, J. (1989) Biochem. J. 262, 939-946 Chasseaud, L. F. (1979) Adv. Cancer Res. 20, 175-293 Del Boccio, G., Di Ilio, C., Miranda, M., Manilla, A., Zarivi, O., Bonfigli, A. & Federici G. (1987a) Comp. Biochem. Physiol. B 86, 749-753 Del Boccio, G., Di Ilio, C., Alin, P., J6rnvall, H. & Mannervik, B. (1987b) Biochem. J. 244, 21-25 Di Ilio, C., Sacchetta, P., Lo Bello, M., Caccuri, A. M. & Federici, G. (1986a) J. Mol. Cell. Cardiol. 18, 983-991
C. Di Ilio and others Di Ilio, C., Del Boccio, G., Miranda, M., Manilla, A., Zarivi, 0. & Federici, G. (1986b) Comp. Biochem. Physiol. C 83, 9-12 Di Ilio, C., Aceto, A., Piccolomini, R., Allocati, N., Faraone, A., Cellini, L., Ravagnan, G. & Federici, G. (1988a) Biochem. J. 255, 971-975 Di Ilio, C., Aceto, A., Del Boccio, G., Casalone, E., Pennelli, A. & Federici, G. (1988b) Eur. J. Biochem. 171, 491-496 Di Ilio, C., Aceto, A., Piccolomini, R., Allocati, N., Caccuri, A. M., Barra, D. & Federici, G. (1989) FEBS Lett. 250, 57-59 Di Ilio, C., Aceto, A., Piccolomini, R., Allocati, N., Faraone, A., Bucciarelli, T., Barra, D. & Federici, G. (1991) Biochim. Biophys. Acta 1077, 141-146 Greenfield, N. & Fasman, G. D. (1969) Biochemistry 8, 4108-4116 Habig, W. H. & Jakoby, W. B. (1981) Methods Enzymol. 77, 398-405 Hayes, J. D. & Wolf, C. R. (1988) in Glutathione Conjugation: Mechanism and Biological Significance (Sies, H. & Ketterer, B., eds.), pp. 315-355, Academic Press, London Jakoby, W. B. & Habig, W. H. (1980) in Enzymatic Basis of Detoxification (Jakoby, W. B., ed.), vol. 2, pp. 63-94, Academic Press, New York Ketterer, B., Meyer, D. J. & Clark, A. G. (1988) in Glutathione Conjugation: Mechanism and Biological Significance (Sies, H. & Ketterer, B., eds.), pp. 73-135, Academic Press, London Laemmli, U. K. (1970) Nature (London) 227, 680-685 Lamoureux, G. L. & Rusness, D. G. (1989) in Glutathione: Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulsen, R. & Avramovic, O., eds.), part B, pp. 153-196, Academic Press, New York Mannervik, B. (1985) Adv. Enzymol. Relat. Areas Mol. Biol. 57, 357-417 Mannervik, B. & Danielson, U. H. (1988) CRC Crit. Rev. Biochem. 23, 281-334 Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Thair, M. K. B., Warholm, M. & J6rnvall, H. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7202-7206 Miranda, M., Di Ilio, C., Del Boccio, G., Bonfigli, A., Zarivi, O., Cimini, A. M., Poma, A. & Amicaralli, F. (1987) Acta Embryol. Morphol. Exp. 8, 293-297 Ostlund Farrants, A. K., Meyer, D. J., Coles, B., Southan, C., Aitken, A., Johnson, P. J. & Ketterer, B. (1987) Biochem. J. 245, 423-428 Piccolomini, R., Di Ilio, C., Aceto, A., Allocati, N., Faraone, A., Cellini, L., Ravagnan, G. & Federici, G. (1989) J. Gen. Microbiol. 135, 3119-3125 Rugh, R. (1961) Experimental Embryology, pp. 56-90, Burges, Minneapolis Simons, P. C. & Vander Jagt, D. L. (1977) Anal. Biochem. 82, 334-341 Sohal, R. S., Allen, R. C. & Nations, C. (1988) Ann. N. Y. Acad. Sci. 551, 59-64 Stanley, J. S. & Benson, A. M. (1988) Biochem. J. 256, 303-306 Tahir, M. K. & Mannervik, B. (1986) J. Biol. Chem. 261, 1048-1051 Towbin, H., Staehelin, T. & Gordon, L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354
Received 16 May 1991/30 August; accepted 12 September 1991
1992