- Email: [email protected]
Cereal dietary proteins with sites for cross-linking by transglutaminase
Vol. 29, No. 9, pp. 2801&2804, 1990. Printed in Great Britain.
0031-9422/90%3.00+0.00 Pergamon Press plc
Phytochemistry,
CEREAL DIETARY PROTEINS WITH SITES FOR CROSS-LINKING TRANSGLUTAMINASE
BY
RAFFAELE PORTA,* VITTORIO GENTILE, CARLA ESPOSITO, LOREDANA MARINIELLO and SALVATORE AURlCCHlot Department of Biochemistry and Biophysics; TDepartment of Pediatrics, University of Naples, Italy (Received
Key Word Index-Cereals;
in revised form 15 February
1990)
spermidine; transglutaminase; A-gliadin.
Abstract-The ability of several cereal proteins to act as substrates for transglutaminase purified from guinea pig liver was investigated. Among the various dietary proteins tested, wheat glutelins and gliadins, as well as purified A-gliadin, were found to be the most effective acyl donor substrates for transglutaminase. In particular, these proteins seemed to be able to produce not only y(glutamy1) spermidine adducts but also polymeric complexes, probably through intermolecular &(y-glutamyl)lysine crosslinks. In the case of A-gliadin, the single lysyl residue occurring in the amino acid sequence (Lys-186) is supposed to act as acyl acceptor site. The peptic-tryptic fragments of gliadins and prolamines of different origin behaved as transglutaminase substrates similarly to native gliadin, mostly in giving rise to large M, polymers. These results are consistent with the hypothesis that animal transglutaminase may be involved in the metabolism of cereal dietary proteins, or of their peptide fragments, when present either in the intestinal lumen or in the mucosal cells.
INTRODUCTION Transglutaminases (TGase, EC 2.3.2.13) are Ca’+-dependent enzymes catalysing an acyl transfer reaction between y-carboxamide groups of peptide-bound glutamine residues and E-amino groups of peptide-bound lysines, or certain primary amines such as histamine, putrescine, spermidine (Spd) and spermine [l-3]. TGase is widely distributed in various animal cells, tissues and body fluids, even though the biological functions of the enzyme have not been fully elucidated [l-S]. Evidence has also been reported [9] indicating that an enzyme with characteristics similar to those found in most TGases, occurs in plant meristematic tissues. Recently it has been demonstrated that there is a significant TGase activity in both rat and human small intestine [lo, 111, whereas a variety of dietary proteins, such as casein, soybean globulins and gliadin, were shown to be able to act in I;itro as acyl donor substrates for the enzyme [12, 131. In addition, the TGase-mediated incorporation of both amino acid derivatives and lysyl dipeptides into some food proteins has been obtained in vitro and proposed as possible and useful tool to improve their functional properties and nutritive values [14, 151. Both gliadins and their proteolytic fragments have been recently demonstrated to play a role in the pathogenesis of gluten-sensitive enteropathy [16]. It has been also reported that an increased TGase activity was observed in jejunal biopsies from patients with untreated coeliac disease compared with control subjects [12]. A possible role of intestinal TGase in gliadin binding to tissues and in the pathogenesis of coeliac disease was postulated [ 12, 161.
*Author to whom correspondence
should
The present paper describes our investigations on the reaction products obtained by treating some cereal proteins (including native and proteolyzed gliadins) with purified preparations of TGase purified from guinea pig liver. RESULTS AND
DISCUSSION
Cereal proteins act as acyl donor substratesfor
TGase
Different dietary proteins, i.e. wheat globulins, glutelins and gliadins, and prolamines from oat, maize and rice, were assayed in vitro as acyl donor substrates for purified liver TGase by using radioactive Spd as acyl acceptor. The fluorography of the gel obtained by subjecting the different assay mixtures to SDS-PAGE at the end of incubation is shown in Fig. 1. The absence of radioactivity in the lanes corresponding to samples incubated without either proteins (lane 7), enzyme (lane 8), or Ca2+ (lane 9) indicates that the radioactive protein bands occurring in lanes l-6 are due to a TGase-catalysed incorporation of [14C]Spd into the different cereal proteins. From the analysis of the intensity of the signals it is evident that wheat gliadins and glutelins are effective acyl donor substrates among the tested proteins. Moreover, the presence of radioactive bands at the top of the gel, detectable with all the cereal proteins, suggests the possible formation of large M, homo- and/or hetero-polymers due to &(y-glutamyl) lysine crosslinks and/or (yglutamyl) polyamine bonds. A-Gliadin
contains
acyl donor and acceptor
sites
From the experiment shown in Fig. 2 (lanes 1-7, A) it is clear that purified A-gliadin is an effective acyl donor substrate for TGase which seems able to produce the
be addressed. 2801
R. PORTA er ul.
2802 1
8
234567
9
Upper Gel Kd - 67
- 25
-
12.3
Fig. 1. TGase-dependent incorporation of Spd into different cereal prokim. The proteins (50 pg) were incubated at 37” for 2 hr in 50 mM TrissHCl butler, pH 8, with 2 pg of purified guinea pig liver TGase. 28 PM L”C]Spd in the presence (lanes I-8) or absence (lane 9) of 3 mM CaCI, (final vol., 60 ~1): lane 1. wheat globulins; lane 2, wheat glutelins; lanes 3, 8 and 9, wheat gliadins; lane 4, oat prolamines: lane 5 maize prolamines: lane 6, rice prolammes. Additional blanks were run simultaneously in the absence of either cereal proteins (lane 7) or enzyme (lane 81. The reactions were stopped by EGTA addition and the samples analysed by SDS-PAGE. The radioactive products were visualized by Ruorography of the gel
A 11234 UI
B 56
mu
7111 illi@I,
2
3
4
5
6
7l8
1, v’;;PW Gel -
Kd -
67
-25
-
12.3
Fig. 2. Inhibition of TGase-dependent incorporation of Spd into wheat gliadins and purified A-gliadin by MDC. 50 fig of either A-gliadin (A) or wheat gliadin mixture (B) were incubated at 37’ for 10 min (lanes I-3) or 120 min (lanes (17), under the same conditions described in Fig. 1, in the absence (lanes 2,4, 7.8) or presence of 1 pM (lanes 1, 5) or 1 mM (lanes 3,6) MDC. Blanks without either CaCI, (lane 7) or enzyme (lane 8) were simultaneously run. The reactions were stopped by EGTA addition and the samples analysed by SDS-PAGE. The radioactive products were visualized by fluorography of the gel.
Transglutaminase
2803
and cereal proteins
dimer and other polymeric forms of the protein (even of 123456789 very large M,) in the presence of Spd. From lanes 3 and 6 it is also evident that unlabelled monodansylcadaverine f Upper (MDC), a well known acyl acceptor substrate of the Gel enzyme, effectively antagonizes the incorporation of radioactive Spd into purified A-gliadin (lanes A) as well -_ as into the proteins contained in the wheat gliadin mixture (lanes B). The inhibition by MDC confirms, indeed, Kd the TGase involvement in the polyamine binding to the -67 _” gliadins. In order to determine if gliadins are also able to give rise to polymers in the absence of a bifunctional acyl acceptor substrate such as Spd, the ability of glycine ethylester (GEE) as an amino donor was tested. The results shown in Fig. 3 indicate that GEE was actively incorporated into both purified A-gliadin (lanes 4 and 5) and proteins contained in the wheat gliadin mixture (lanes 7 and 8), and that MDC was also able to antagonize the GEE binding (lanes 6 and 9). Unexpectedly, labelled bands corresponding to proteins of larger M, appeared in lanes 5 and 8, where samples incubated 2 hr with TGase were run. As GEE is endowed with only one primary -12.3 amino group, E-amino groups of endoprotein lysyl residues were probably involved in intermolecular c(YFig. 3. TGase-dependent incorporation of GEE into wheat glutamyl)lysine crosslinks responsible for the formation gliadins and purified A-gliadin and inhibitory effect of MDC. of gliadin polymers. This result suggests, in the case of 50 ng of either A-gliadin (lanes 2-6) or wheat gliadin mixture A-gliadin, that the single lysine (Lys-186) occurring in the (lanes 7-9) were incubated with 28 PM [‘%]GEE at 37” for 10 protein sequence [17] is able to act as acyl acceptor min (lanes 4, 7) or 120 min (lanes l-3, 5,6, 8,9), under the same substrate of TGase. Gliadin and proiamine peptidic fragments act as acyl donor substrates for TGase
In order to test if the proteolytic digestion modifies the ability of gliadins and prolamines to act as TGase sub-
conditions described in Fig. 1, in the absence (lanes 1-5, 7, 8) or presence (lanes 6, 9) of 1 mM MDC. Blanks without either acyl donor substrate (lane l), enzyme (lane 2) or CaCl, (lane 3) were simultaneously run. The reactions were stopped by EGTA addition and the samples analysed by SDS-PAGE. The radioactive products were visualized by fluorography of the gel.
B
A 11
2
3
4
5
6’
‘1
2
3
4
5
6’
Kd -67
-25
-
12.3
Fig. 4. TGase-dependent incorporation of Spd (A) or GEE (B) into peptide fragments derived from enzymatic digestion of gliadins and prolamines. 100 ng of peptide mixtures obtained from peptic-tryptic hydrolysis (see text) of gliadins from S. Pastore wheat (lane 1) and Rieti wheat (lane 2), and of prolamines from barley (lane 3), oats (lane 4), and rye (lane 5), were incubated at 37” for 2 hr under the same conditions described in Fig. 1. Blanks without peptides (lane 6) were simultaneously run. The reactions were stopped by EGTA addition and the samples analysed by SDS-PAGE. The radioactive products were visualized by fluorography of the gels.
2804
R. PORTA et al.
strates, proteins of different origin (S. Pastore and Rieti wheat gliadins, and barley, oats and rye prolamines) were separately subjected to a peptic-tryptic hydrolysis and the resulting peptide fragments assayed as acyl donors for liver TGase. All the peptide mixtures, tested by using [14C]Spd (lanes A) or [14C]GEE (lanes B) as acyl acceptors, gave rise to marked radioactive signals on the gels after SDS-PAGE and fluorography (Fig. 4). It is also evident from the gel that Spd was more effective in the labelling of the peptides in comparison with GEE, and that fragments contained in all the proteolysates of gliadins and prolamines of different origin had the ability to produce large M, polymers. Furthermore, the TGasecatalysed formation of these polymers in the presence of GEE seems to confirm the data discussed above on the existence, in the gliadin polypeptide chain, of a reactive lysyl residue able to act as an acyl acceptor site.
EXPERIMENTAL Materials. Spd trihydrochloride and MDC were purchased from Sigma. [‘4C]Spd trihydrochloride (118 mCi mmol-‘) was obtained from Amersham U.K. (glycine-l-[‘4C])GEE hydrochloride (60 mCi mmol- ‘) was from New England Nuclear, F.R.G. Acrylamide and methylene-bisacrylamide were from Fluka. Cytochrome c, chymotrypsinogen A, and bovine serum albumin were from Serva. Dithiothreitol was from CalbiochemBehring. TGase was purified to homogeneity from guinea pig liver according to ref. [IS]. Wheat gliadins, glutelins and globulins, oat prolamines, maize prolamines, rice prolamines, as well as the tryptic-peptic digests of S. Pastore or Rieti wheat gliadins, and of barley, oats and rye prolamines, were generous gifts of Prof. V. Silano and Dr. M. De Vincenzi (Istituto Superiore di Sanita’, Roma). A-Gliadin [17] was a generous gift of Dr D. Kasarda (Food Proteins Research Unit, Western Regional Center, Agricultural Research Service, U.S. Department of Agriculture, Berkeley, CA 94710). All other chemicals were the purest available grades from standard commercial sources. Assay of TGase activity. TGase activity was assayed by a radiometric method based on the Ca2+-dependent incorporation of isotopically labelled Spd or GEE into acyl donor protein substrates [3]. Incubation was carried out at 37” and blanks run simultaneously without either CaZ+, enzyme source or protein substrate, or in the presence of EGTA. At the end of incubation the reaction was stopped by IO mM EGTA (final concn). The visualization of the radioactive Spd or GEE bound to the cereal
proteins was achieved by SDS-(lS%)PAGE and fluorography [19, 201. The composition of the reaction mixtures and further experimental details are specifically described in the legends to the figures of the single experiments. Protein determination. Protein concentration was determined by the method of ref. [21]. Acknowledgement-This work has been supported from Minister0 della Pubblica Istruzione, Italy.
by grants
REFERENCES 1. Williams-Ashman, H. G. and Canellakis, Z. N. (1980) Physiol. Chem. Physics. 12, 457. 2. Lorand, I,. and Conrad, S. M. (1984) Mol. Cell. Biochem. 58, 9. 3. Folk, .I. E. and Chung, S. I. (1985) Meth. Enzymol. 113, 358. 4. Folk, J. E. and Chung, S. I. (1973) Ado. Enzymol. 38. 109. 5. Folk, J. E. (1980) Ann. Rev. Biochem. 49. 517. 6. Lorand, L., Credo. R. B.. and Janus. T. J. (1981) Meth. Enzymol. 80, 333. 7. Fesus, L., Harsfalvi, J.. Horvath, A. and Sandor, M. (1984) Mol. Immunol. 21, 1161. 8. Williams-Ashman, H. G. (1984) Mol. Cell. Biochem. 58, 51. 9. Icekson, I. and Apelbaum, A. (1987) Plant Phjssiol. 84, 972. 10. Pate], E. K., Bruce, S. E., Bjamason. I. and Peters, 7. J. (1985) Cell. B&hem. Fwwt. 3, 199. 11. D’Argenio, G., Sorrentini, I., Ciacci, C. and Mazzacca, G. (1988) Enzyme 39, 227. 12. Bruce, S. E., Bjarnason, I. and Peters. T. J. (1985) C[in. Sci. 68, 573. K. (1984) Agric. Biol. 13. Motoki, M., Nio. N. and Takinami, Chem. 48, 1257. 14. Ikura, K., Goto, M., Yoshikawa, M., Sasaki. R. and Chiba, H. (1984) Agric. Biol. Chem. 48, 2347. 15. Ikura, K., Okumura, K.. Yoshikawa, M.. Sasaki, R. and Chiba, H. (1985) Agric. Biol. Chem. 49. 1877. 1. (1984) Gut 25, 913. 16. Peters, T. J. and Bjamason, 17. Kasarda, D. D., Okita, T. W., Bernardin, J. E. and Baeker, P. A. (1984) Proc. Nat1 Acad. Sci. U.S.A. 81, 4712. 18. Connellan, J. M.. Chung. S. I., Whetzel. N. K., Bradley, L. M. and Folk, J. E. (1971) J. Biol. Chem. 246, 1093. 19. Laemmli, U. K. (1970) Nature 277, 680. 20. Banner. W. M. and Laskey. R. A. (1974) Eur. J. Biochem. 46, 83. 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193. 265.
0031-9422/90%3.00+0.00 Pergamon Press plc
Phytochemistry,
CEREAL DIETARY PROTEINS WITH SITES FOR CROSS-LINKING TRANSGLUTAMINASE
BY
RAFFAELE PORTA,* VITTORIO GENTILE, CARLA ESPOSITO, LOREDANA MARINIELLO and SALVATORE AURlCCHlot Department of Biochemistry and Biophysics; TDepartment of Pediatrics, University of Naples, Italy (Received
Key Word Index-Cereals;
in revised form 15 February
1990)
spermidine; transglutaminase; A-gliadin.
Abstract-The ability of several cereal proteins to act as substrates for transglutaminase purified from guinea pig liver was investigated. Among the various dietary proteins tested, wheat glutelins and gliadins, as well as purified A-gliadin, were found to be the most effective acyl donor substrates for transglutaminase. In particular, these proteins seemed to be able to produce not only y(glutamy1) spermidine adducts but also polymeric complexes, probably through intermolecular &(y-glutamyl)lysine crosslinks. In the case of A-gliadin, the single lysyl residue occurring in the amino acid sequence (Lys-186) is supposed to act as acyl acceptor site. The peptic-tryptic fragments of gliadins and prolamines of different origin behaved as transglutaminase substrates similarly to native gliadin, mostly in giving rise to large M, polymers. These results are consistent with the hypothesis that animal transglutaminase may be involved in the metabolism of cereal dietary proteins, or of their peptide fragments, when present either in the intestinal lumen or in the mucosal cells.
INTRODUCTION Transglutaminases (TGase, EC 2.3.2.13) are Ca’+-dependent enzymes catalysing an acyl transfer reaction between y-carboxamide groups of peptide-bound glutamine residues and E-amino groups of peptide-bound lysines, or certain primary amines such as histamine, putrescine, spermidine (Spd) and spermine [l-3]. TGase is widely distributed in various animal cells, tissues and body fluids, even though the biological functions of the enzyme have not been fully elucidated [l-S]. Evidence has also been reported [9] indicating that an enzyme with characteristics similar to those found in most TGases, occurs in plant meristematic tissues. Recently it has been demonstrated that there is a significant TGase activity in both rat and human small intestine [lo, 111, whereas a variety of dietary proteins, such as casein, soybean globulins and gliadin, were shown to be able to act in I;itro as acyl donor substrates for the enzyme [12, 131. In addition, the TGase-mediated incorporation of both amino acid derivatives and lysyl dipeptides into some food proteins has been obtained in vitro and proposed as possible and useful tool to improve their functional properties and nutritive values [14, 151. Both gliadins and their proteolytic fragments have been recently demonstrated to play a role in the pathogenesis of gluten-sensitive enteropathy [16]. It has been also reported that an increased TGase activity was observed in jejunal biopsies from patients with untreated coeliac disease compared with control subjects [12]. A possible role of intestinal TGase in gliadin binding to tissues and in the pathogenesis of coeliac disease was postulated [ 12, 161.
*Author to whom correspondence
should
The present paper describes our investigations on the reaction products obtained by treating some cereal proteins (including native and proteolyzed gliadins) with purified preparations of TGase purified from guinea pig liver. RESULTS AND
DISCUSSION
Cereal proteins act as acyl donor substratesfor
TGase
Different dietary proteins, i.e. wheat globulins, glutelins and gliadins, and prolamines from oat, maize and rice, were assayed in vitro as acyl donor substrates for purified liver TGase by using radioactive Spd as acyl acceptor. The fluorography of the gel obtained by subjecting the different assay mixtures to SDS-PAGE at the end of incubation is shown in Fig. 1. The absence of radioactivity in the lanes corresponding to samples incubated without either proteins (lane 7), enzyme (lane 8), or Ca2+ (lane 9) indicates that the radioactive protein bands occurring in lanes l-6 are due to a TGase-catalysed incorporation of [14C]Spd into the different cereal proteins. From the analysis of the intensity of the signals it is evident that wheat gliadins and glutelins are effective acyl donor substrates among the tested proteins. Moreover, the presence of radioactive bands at the top of the gel, detectable with all the cereal proteins, suggests the possible formation of large M, homo- and/or hetero-polymers due to &(y-glutamyl) lysine crosslinks and/or (yglutamyl) polyamine bonds. A-Gliadin
contains
acyl donor and acceptor
sites
From the experiment shown in Fig. 2 (lanes 1-7, A) it is clear that purified A-gliadin is an effective acyl donor substrate for TGase which seems able to produce the
be addressed. 2801
R. PORTA er ul.
2802 1
8
234567
9
Upper Gel Kd - 67
- 25
-
12.3
Fig. 1. TGase-dependent incorporation of Spd into different cereal prokim. The proteins (50 pg) were incubated at 37” for 2 hr in 50 mM TrissHCl butler, pH 8, with 2 pg of purified guinea pig liver TGase. 28 PM L”C]Spd in the presence (lanes I-8) or absence (lane 9) of 3 mM CaCI, (final vol., 60 ~1): lane 1. wheat globulins; lane 2, wheat glutelins; lanes 3, 8 and 9, wheat gliadins; lane 4, oat prolamines: lane 5 maize prolamines: lane 6, rice prolammes. Additional blanks were run simultaneously in the absence of either cereal proteins (lane 7) or enzyme (lane 81. The reactions were stopped by EGTA addition and the samples analysed by SDS-PAGE. The radioactive products were visualized by Ruorography of the gel
A 11234 UI
B 56
mu
7111 illi@I,
2
3
4
5
6
7l8
1, v’;;PW Gel -
Kd -
67
-25
-
12.3
Fig. 2. Inhibition of TGase-dependent incorporation of Spd into wheat gliadins and purified A-gliadin by MDC. 50 fig of either A-gliadin (A) or wheat gliadin mixture (B) were incubated at 37’ for 10 min (lanes I-3) or 120 min (lanes (17), under the same conditions described in Fig. 1, in the absence (lanes 2,4, 7.8) or presence of 1 pM (lanes 1, 5) or 1 mM (lanes 3,6) MDC. Blanks without either CaCI, (lane 7) or enzyme (lane 8) were simultaneously run. The reactions were stopped by EGTA addition and the samples analysed by SDS-PAGE. The radioactive products were visualized by fluorography of the gel.
Transglutaminase
2803
and cereal proteins
dimer and other polymeric forms of the protein (even of 123456789 very large M,) in the presence of Spd. From lanes 3 and 6 it is also evident that unlabelled monodansylcadaverine f Upper (MDC), a well known acyl acceptor substrate of the Gel enzyme, effectively antagonizes the incorporation of radioactive Spd into purified A-gliadin (lanes A) as well -_ as into the proteins contained in the wheat gliadin mixture (lanes B). The inhibition by MDC confirms, indeed, Kd the TGase involvement in the polyamine binding to the -67 _” gliadins. In order to determine if gliadins are also able to give rise to polymers in the absence of a bifunctional acyl acceptor substrate such as Spd, the ability of glycine ethylester (GEE) as an amino donor was tested. The results shown in Fig. 3 indicate that GEE was actively incorporated into both purified A-gliadin (lanes 4 and 5) and proteins contained in the wheat gliadin mixture (lanes 7 and 8), and that MDC was also able to antagonize the GEE binding (lanes 6 and 9). Unexpectedly, labelled bands corresponding to proteins of larger M, appeared in lanes 5 and 8, where samples incubated 2 hr with TGase were run. As GEE is endowed with only one primary -12.3 amino group, E-amino groups of endoprotein lysyl residues were probably involved in intermolecular c(YFig. 3. TGase-dependent incorporation of GEE into wheat glutamyl)lysine crosslinks responsible for the formation gliadins and purified A-gliadin and inhibitory effect of MDC. of gliadin polymers. This result suggests, in the case of 50 ng of either A-gliadin (lanes 2-6) or wheat gliadin mixture A-gliadin, that the single lysine (Lys-186) occurring in the (lanes 7-9) were incubated with 28 PM [‘%]GEE at 37” for 10 protein sequence [17] is able to act as acyl acceptor min (lanes 4, 7) or 120 min (lanes l-3, 5,6, 8,9), under the same substrate of TGase. Gliadin and proiamine peptidic fragments act as acyl donor substrates for TGase
In order to test if the proteolytic digestion modifies the ability of gliadins and prolamines to act as TGase sub-
conditions described in Fig. 1, in the absence (lanes 1-5, 7, 8) or presence (lanes 6, 9) of 1 mM MDC. Blanks without either acyl donor substrate (lane l), enzyme (lane 2) or CaCl, (lane 3) were simultaneously run. The reactions were stopped by EGTA addition and the samples analysed by SDS-PAGE. The radioactive products were visualized by fluorography of the gel.
B
A 11
2
3
4
5
6’
‘1
2
3
4
5
6’
Kd -67
-25
-
12.3
Fig. 4. TGase-dependent incorporation of Spd (A) or GEE (B) into peptide fragments derived from enzymatic digestion of gliadins and prolamines. 100 ng of peptide mixtures obtained from peptic-tryptic hydrolysis (see text) of gliadins from S. Pastore wheat (lane 1) and Rieti wheat (lane 2), and of prolamines from barley (lane 3), oats (lane 4), and rye (lane 5), were incubated at 37” for 2 hr under the same conditions described in Fig. 1. Blanks without peptides (lane 6) were simultaneously run. The reactions were stopped by EGTA addition and the samples analysed by SDS-PAGE. The radioactive products were visualized by fluorography of the gels.
2804
R. PORTA et al.
strates, proteins of different origin (S. Pastore and Rieti wheat gliadins, and barley, oats and rye prolamines) were separately subjected to a peptic-tryptic hydrolysis and the resulting peptide fragments assayed as acyl donors for liver TGase. All the peptide mixtures, tested by using [14C]Spd (lanes A) or [14C]GEE (lanes B) as acyl acceptors, gave rise to marked radioactive signals on the gels after SDS-PAGE and fluorography (Fig. 4). It is also evident from the gel that Spd was more effective in the labelling of the peptides in comparison with GEE, and that fragments contained in all the proteolysates of gliadins and prolamines of different origin had the ability to produce large M, polymers. Furthermore, the TGasecatalysed formation of these polymers in the presence of GEE seems to confirm the data discussed above on the existence, in the gliadin polypeptide chain, of a reactive lysyl residue able to act as an acyl acceptor site.
EXPERIMENTAL Materials. Spd trihydrochloride and MDC were purchased from Sigma. [‘4C]Spd trihydrochloride (118 mCi mmol-‘) was obtained from Amersham U.K. (glycine-l-[‘4C])GEE hydrochloride (60 mCi mmol- ‘) was from New England Nuclear, F.R.G. Acrylamide and methylene-bisacrylamide were from Fluka. Cytochrome c, chymotrypsinogen A, and bovine serum albumin were from Serva. Dithiothreitol was from CalbiochemBehring. TGase was purified to homogeneity from guinea pig liver according to ref. [IS]. Wheat gliadins, glutelins and globulins, oat prolamines, maize prolamines, rice prolamines, as well as the tryptic-peptic digests of S. Pastore or Rieti wheat gliadins, and of barley, oats and rye prolamines, were generous gifts of Prof. V. Silano and Dr. M. De Vincenzi (Istituto Superiore di Sanita’, Roma). A-Gliadin [17] was a generous gift of Dr D. Kasarda (Food Proteins Research Unit, Western Regional Center, Agricultural Research Service, U.S. Department of Agriculture, Berkeley, CA 94710). All other chemicals were the purest available grades from standard commercial sources. Assay of TGase activity. TGase activity was assayed by a radiometric method based on the Ca2+-dependent incorporation of isotopically labelled Spd or GEE into acyl donor protein substrates [3]. Incubation was carried out at 37” and blanks run simultaneously without either CaZ+, enzyme source or protein substrate, or in the presence of EGTA. At the end of incubation the reaction was stopped by IO mM EGTA (final concn). The visualization of the radioactive Spd or GEE bound to the cereal
proteins was achieved by SDS-(lS%)PAGE and fluorography [19, 201. The composition of the reaction mixtures and further experimental details are specifically described in the legends to the figures of the single experiments. Protein determination. Protein concentration was determined by the method of ref. [21]. Acknowledgement-This work has been supported from Minister0 della Pubblica Istruzione, Italy.
by grants
REFERENCES 1. Williams-Ashman, H. G. and Canellakis, Z. N. (1980) Physiol. Chem. Physics. 12, 457. 2. Lorand, I,. and Conrad, S. M. (1984) Mol. Cell. Biochem. 58, 9. 3. Folk, .I. E. and Chung, S. I. (1985) Meth. Enzymol. 113, 358. 4. Folk, J. E. and Chung, S. I. (1973) Ado. Enzymol. 38. 109. 5. Folk, J. E. (1980) Ann. Rev. Biochem. 49. 517. 6. Lorand, L., Credo. R. B.. and Janus. T. J. (1981) Meth. Enzymol. 80, 333. 7. Fesus, L., Harsfalvi, J.. Horvath, A. and Sandor, M. (1984) Mol. Immunol. 21, 1161. 8. Williams-Ashman, H. G. (1984) Mol. Cell. Biochem. 58, 51. 9. Icekson, I. and Apelbaum, A. (1987) Plant Phjssiol. 84, 972. 10. Pate], E. K., Bruce, S. E., Bjamason. I. and Peters, 7. J. (1985) Cell. B&hem. Fwwt. 3, 199. 11. D’Argenio, G., Sorrentini, I., Ciacci, C. and Mazzacca, G. (1988) Enzyme 39, 227. 12. Bruce, S. E., Bjarnason, I. and Peters. T. J. (1985) C[in. Sci. 68, 573. K. (1984) Agric. Biol. 13. Motoki, M., Nio. N. and Takinami, Chem. 48, 1257. 14. Ikura, K., Goto, M., Yoshikawa, M., Sasaki. R. and Chiba, H. (1984) Agric. Biol. Chem. 48, 2347. 15. Ikura, K., Okumura, K.. Yoshikawa, M.. Sasaki, R. and Chiba, H. (1985) Agric. Biol. Chem. 49. 1877. 1. (1984) Gut 25, 913. 16. Peters, T. J. and Bjamason, 17. Kasarda, D. D., Okita, T. W., Bernardin, J. E. and Baeker, P. A. (1984) Proc. Nat1 Acad. Sci. U.S.A. 81, 4712. 18. Connellan, J. M.. Chung. S. I., Whetzel. N. K., Bradley, L. M. and Folk, J. E. (1971) J. Biol. Chem. 246, 1093. 19. Laemmli, U. K. (1970) Nature 277, 680. 20. Banner. W. M. and Laskey. R. A. (1974) Eur. J. Biochem. 46, 83. 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193. 265.