ANALYTICAL
BIOCHEMISTRY
Preparation
88, 587-597 (1978)
and Characterization of Biologically Active lodo- and C3H]Secretin
JIANN-TRZUO LIN, SLOBODAN MILUTINOVIC, AND FALKFAHRENHOLZ Max-Planck-Institut
ftir
Biopltysik,
FrankfurtlMain,
Germany
Received October 14, 1977; accepted March 17. 1978 Synthetic secretin has been iodinated at the N-terminal histidine, leading to an almost 100% yield of mono- and diiodo-secretin (“iodo-secretin”). The catalytic exchange of iodine against tritium results in the preparation of secretin labeled with tritium mainly at the hktidine residue (7 Ciimmol). Iodo-secretin and [YH]secretin have the same potency in stimulating pancreatic adenylate cyclase as secretin, but the apparent affinity of [3H]secretin for this enzyme is twice as high as for iodo-secretin. [3H]Secretin binds rapidly to pancreatic plasma membranes. Adding excess unlabeled secretin reduces the tracer binding by about 70%.
It is well known that the peptide hormone secretin regulates electrolyte and water secretion of the pancreas (1). Conclusions about the nature of its receptor have been deduced indirectly from structure activity studies by Bodanszky (2) and Wunsch et al. (3). The interaction of rz51-labeled secretin with pancreatic plasma membranes was studied by Milutinovic ef al. (4), and the results were interpreted to mean that secretin and 1251labeled secretin have high affinity to the same binding site. The labeled derivative, however, which was prepared with 1251Na and chloramine-T according to the method of Hunter and Greenwood (5), in contrast with secretin itself, did not stimulate pancreatic adenylate cyclase. Radioactive labeling of secretin is particularly difficult for want of tyrosine; besides, iodination at the N-terminal histidine is determined by the low reactivity of the imidazole nucleus (6). Introduction of iodine into secretin by the chloramine-T method (7,8) or the lactoperoxidase method (9) lead to a mixture of labeled and unlabeled secretin with high specific radioactivity used in radioimmunoassay studies. A higher yield of iodinated peptides but with a much lower specific radioactivity can be obtained by using iodine monochloride (ICl). There is evidence that incorporation of one atom of iodine per molecule of secretin by ICI causes a two- to threefold decrease in the apparent affinity toward pancreatic adenylcyclase (10). The introduction of the large iodine atom into the N-terminal histidine, which is indispensabie for full biological 587
0003-2697/78/0882-0587$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction m any form reserved.
588
LIN,
MILUTINOVIC,
AND
FAHRENHOLZ
activity of secretin (1 l), might also affect the affinity of the labeled hormone to its receptor. This, in turn, could lead to nonlinear Scatchard plots of binding data (12). These difficulties may be avoided by using tritiated secretin in binding experiments. Polypeptides can be labeled with tritium on the tyrosine or histidine by catalytic exchange reaction of iodine against tritium (13,14). This paper describes the preparation of biologically active [3H]secretin (labeled at the histidine residue via the iodo intermediate), suitable for a study of the interaction between the hormone and its receptor. MATERIALS
AND METHODS
Synthetic secretin acetate was a gift by Prof. E. Wiinsch, Max-Planck Institute of Biochemistry (Munich, Germany). The dipeptide histidylserine, acrylamide, N,N’-methylenebisacrylamide, and ammonium persulfate were purchased from Serva (Heidelberg, Germany); tritium gas, (a- 32P)labeled adenosine 5’-triphosphate (2-10 Ci/mmol, sodium salt), and [83H]adenosine-3’-5’-cyclic phosphate (27 Ci/mmol) were from Amersham Buchler (Braunschweig, Germany); soybean tripsin inhibitor, pyruvate kinase (crystalline suspension, 10 mg/ml), and phosphoenolpyruvate (trisodium salt) were from Boehringer (Mannheim, German); bacitracin and albumin fraction V were from Sigma; EHWPO filters (pore size, 0.5 pm) were from the Millipore Corp.; the catalyst 10% Pd/C and aminopeptidase M were from Carl Roth (Karlsruhe, Germany); Sephadex G-25 and Sephadex SP-C25 were from Phamacia Fine Chemicals (Sweden); 1251C1(50mCi/ mmol) was obtained from New England Nuclear (Boston, Massachusetts); ICI was from Merck-Schuchard (Darmstadt, Germany); and Whatman 3 MM paper for electrophoresis was from Whatman Biochemicals Ltd. (Maidston, England). The catalyst 10% Pd/C was activated at 120°C for 24 hr under vacuum. Unlabeled ICI was sublimed twice at 20°C under vacuum and stored in dry dioxane at 4°C. The ICI content was determined by titration with sodium thiosulfate. Amino acid analyses were performed with a system LC 6000 from Biotronik, Wissenschaftliche Gerlte GmbH, Germany. High voltage electrophoresis was carried out with the Pherograph Mini 65 from Hormuth-Vetter (Wiesloch, Germany); ultraviolet absorption spectra were recorded with the spectrophotometer, type DB-GT, from Beckman Instruments; radioactivity was measured with the autogammascintillation spectrometer, model 5100, from Packard, and the betascintillation spectrometer, model BF 5000, was from Friske & Horpfner GmbH (Karlsruhe, Germany). Column effluents were monitored with an Uvicord III from LKB (Sweden) at wavelengths of 205 and 256 nm. For monitoring at a wavelength of 220 nm, the spectrophotometer DB-GT was used with a flow cell of 300 ~1. Zodination of secretin. Secretin, 3 pmol, was dissolved in 0.5 ml of
SECRETIN
PREPARATION
AND ISOLATION
589
guanidine hydrochloride (7 M) and diluted with 2.5 ml of 0.2 M ammonium acetate/ammonia solution (PH 9.2), which had been cooled to 4°C. The solution was stirred vigorously in an ice bath, and 9 pmol of iodine monochloride containing lz51Cl as tracer (about lo6 cpm) in 80 ~1 of dry dioxane was added dropwise to the solution within 20 sec. After the solution was stirred for 1 min, the reaction was stopped by adding 90 ,ul of a 0.1 M aqueous hydrazinhydrate solution. All iodinated products were protected from light. Isolation and characterization of iodo-secretin. The reaction mixture was lyophilized, redissolved in 1 ml of 0.5% acetic acid, and applied to a Sephadex G-25 column (90 x 1.25 cm) equilibrated with 0.5% acetic acid at 4°C. Eluting was performed with the same medium, and in each fraction (1.8 ml) the radioactivity was measured. The radioactive fractions were lyophilized and examined by high-voltage paper electrophoresis at pH 6.2, by gel electrophoresis according to Ahlroth et al. (15), and by ultraviolet absorption spectroscopy. For detection of peptides on paper, we used the ninhydrin, Pauly, and FFCA’ (16) reagents. Acid hydrolyses for amino acid analysis were performed with 6 N HCl at 110°C for 20 hr in the presence of norleucine as standard. The yield of peptides was estimated by quantitative amino acid analysis of aliquots using glutamic acid or phenylalanine as a measure of the amount of peptides present. For enzymatic hydrolysis, 5 nmol of peptide, dissolved in 100 ~1 of 0.2 M triethylammonium acetate buffer (PH 8.0), was incubated at 37°C with 100 mU of aminopeptidase M dissolved in 50 ~1 of water. After 16 hr the pH was adjusted to be 3.5 by adding 1 N acetic acid and the mixture was evaporated to dryness. Tritiation. After iodination of 1.5 pmol of secretin we immediately added 0.5% acetic acid to the reaction mixture to adjust the pH to 8.3. The solution was kept frozen in liquid nitrogen until we transferred it into a special flask connected to a vacuum line similar to that described by Morgat et al. (13). The solution was frozen, and vacuum was applied to remove all gas which was dissolved. Pd/C catalyst (10% Pd) was then added, and 20 Ci of pure tritium gas was introduced at a pressure of about 500 mm Hg. The mixture was brought to 10°C and stirred magnetically. After an hour the solution was frozen again and tritium gas was removed. By adding 1 N acetic acid, we acidified and simultaneously diluted the mixture to a volume of 10 ml. Centrifugation, followed by repeated lyophilization, gave a white material which was redissolved in 0.5% acetic acid and stored in liquid nitrogen. Zsofation and purijication of [3H]secretin. To remove the rest of labile tritium and inorganic salts, the reaction mixture was filtered on a Sephadex G-25 column (90 x 1.25 cm), equilibrated with 0.5% acetic acid, and eluted 1 Ferric ferricyanide-orsenius
acid.
590
LIN,
MILUTINOVIC,
AND
FAHRENHOLZ
with the same solvent. To examine the eluant, the same procedure was used as for the identification of iodo-secretin. The fractions which contained P-radioactivity were collected and lyophilized. We dissolved the dry material in 0.01 M ammonium acetate buffer @H 6.0) and chromatographed it on a Sephadex SP-C25 column (5 x 0.7 cm) equilibrated with the same buffer. The chromatography was performed at 4”C, as described by Wtinsch et al. (17); ammonium acetate buffer, however, was used instead of ammonium carbonate buffer as the eluant (18). Fractions of 3.7 ml were collected. To determine the [3H]secretin-containing fractions, we applied the same analytical methods as those used for the characterization of iodo-secretin. To find the location of tritium label in the peptide chain, we digested rH]secretin by aminopeptidase M and applied the hydrolyzate to the amino acid analyser. Fractions of the eluant were collected, and the P-radioactivity in each was measured. Preparation of +iodo-L-histidine (MZH] and 2,4-diiodo-L-histidine (DZH). Iodo-histidines were prepared either according to the method of Brunings (19) or of Savoie et al. (20). The position of the iodine atom in
the imidazole ring was determined by nmr spectroscopy of the compounds dissolved in a mixture of DzO/CF3COOD (21). Stimulation of pancreatic adenylate cyclase activity. Pancreatic plasma membranes were prepared as described (4). Adenylate cyclase activity was measured essentially by the method of Krishna et al. (22) with some modifications (4). The following incubation medium was used: 0.8 mM [a32P]ATP, 5 mM MgC12, 40 mM Tris-Cl (PH 7.4), 10 mM theophylline, 80 to 120 pg of membrane protein, and an ATP-regenerating system containing 5 mM phosphoenlpyruvate and 0.2 mg of pyruvate kinase/ml. The final volume was 0.1 ml. Incubations were carried out at 30°C for 20 min and stopped by adding a 1% SDS solution. The apparent affinity of secretin derivatives was expressed as a percentage of the apparent affinity of secretin: (K, of secretin/K, of secretin derivative) x 100. Binding assay of secretin derivatives to pancreatic membranes. Membranes (150 pg of protein) were added to the preequilibrated assay mixture (described in legends to Fig. 7), and after incubation at 22°C for different times, the binding was determined by filtration through a Millipore filter, as described by Milutinovic et al. (4). RESULTS Zodosecretin. In a preliminary experiment we observed that a large excess of ICI or that long reaction times decreased the yield of iodinated dipeptide histidyl-serine (position l-2 of secretin). Therefore, iodination of secretin was carried out with 2.5 to 4 Eq of ICI at 4”C, the reaction being terminated after 1 min. The elution diagram (Fig. 1) of the reaction mixture on Sephadex G-25 shows two peaks of y-radioactivity, the first one
SECRETIN
PREPARATION
591
AND ISOLATION
k IW-
EO-
8 5 z 2 4
60-
LO-
20 -
O-
10
FIG. 1. SephadexG-25 column chromatography of iodinated secretin: 6) uv absorbance at 205 nm; t-a-e-0) uv absorbance at 254 nm; and (----) y-radioactivity. The first peak contains iodo-secretin.
(fractions 20-35) containing all eluted peptides and the second one (fractions S-80) containing iodide and salts. The degree to which secretin is modified can be examined by gel electrophoresis and amino acid analysis of the peptides eluted in the first peak. Only traces of secretin show up in gel electrophoresis studies. After digestion with aminopeptidase M, the amino acid analysis reveals the presence of MIH and DIH (MIH:DIH = 2: 1 for 2.5 Eq of ICI and =l:l for 4 Eq of ICl) instead of histidine. The exact ratio of iodohistidines is difficult to determine, since the hydrolysis of secretin, iodinated at its N-terminal residue with aminopeptidase M, is much slower than that of unmodified secretin. Also, conversion of some DIH to MIH during enzymatic digestion has been reported (20). After hydrolysis of iodinated secretin by 6 N HCl some histidine is found (Phe:His = 1:O.l; Fig. 2), mainly because of the complete destruction of DIH and the partial destruction of MIH (23, 24). From the preceding it is obvious that secretin is iodinated at its N-terminal histidyl residue with a nearly 100% yield, leading to mono- and disubstituted derivatives (iodosecretin). During gel electrophoresis in the system of Ahlroth and Mutt (15) and during high voltage paper electrophoresis at pH 6.5, iodo-secretin migrates toward the cathode more slowly than secretin. Iodo-secretin gives an intense blue color reaction with FFCA reagent and only gives a faint color with Pauly reagent and ninhydrin. Its ultraviolet absorption spectrum in 0.5% acetic acid shows an additional maximum at 274 nm (E = 2140). [3H]Secretin. Iodine can be completely exchanged against tritium and hydrogen by treating an aqueous solution of iodosecretin at pH 8.3 in the presence of 10% Pd/C. After the reaction mixture is filtered by Sephadex G-25, the eluted peptides seem to contain no y-radioactivity. Gel elec-
592
LIN, MILUTINOVIC,
AND FAHRENHOLZ
FIG. 2. Amino acid analysis diagram of iodinated secretin after hydrolysis with 6 N HCl at 11°C for 20 hr. Conditions: column, 45 x 0.6 cm; Durum resin DCdA: temperature, 70°C; eluting buffer, pH 6.35; and 1.4 N Na+.
trophoresis shows one major band corresponding to [3H]secretin and two minor components (Fig. 3); the latter can be removed by ion exchange chromatography with Sephadex SP-C25 (Fig. 4). Amino acid analysis of chemically pure tritiated secretin (fractions 99-153) gives a one-to-one ratio of histidine to phenylalanine (Fig. 5). We find that most of the pradioactivity is incorporated into histidine; however, 15% of the specific radioactivity of histidine is located in the phenylalanyl residue. The specific radioactivity of [3H]secretin is found to be 7 Cilmmol (theoretical: 3060Ci/mmol), and the yield of tritiated secretin is 15% of the initial unlabeled secretin. The results reported above were obtained by tritiation of iodosecretin in ammonium acetate buffer in the presence of an excess of iodide. When the iodide is removed and the reaction is performed in 0.2 M borate buffer @H 8.3), the results remain essentially unchanged.
SECRETIN
PREPARATION
593
AND ISOLATION
FIG. 3. Gel electrophoresis of rYH]secretin in the system described by Ahlroth et al. (15): (A) [3H]secretin before purification; (B) [3H]secretin after purification on Sephadex SP-C25 (fractions 99-153); (C) synthetic secretin; and (D) [3H]secretin plus secretin.
Tritiation of secretin treated for iodination with a large excess of ICI (10 equivalents) for 10 min yields a much lower specific radioactivity. The low ratio of histidine to phenylalanine (0.2: 1) obtained by amino acid analysis of this tritiated material indicates the occurrence of side reactions at the histidyl residue during iodination. Biological activity. Iodo-secretin and [3H]secretin stimulate pancreatic adenylcyclase to the same extent as secretin. The K, values computed graphically from Fig. 6 show that the apparent affinities of iodo-secretin b
105PH
T
C[Ml
E s
lOI-
ij s H 103-
I
102Fraction
0
numba
FIG. 4. Purification of [3H]secretin by chromatography on Sephadex SP-C25: (----) uv absorbance at 220 nm, (-) P-radioactivity, (-e-o-o) concentration of eluting buffer, and (- - -) pH of eluting buffer. Fractions 99 to 153 contain [3H]secretin.
594
LIN, MILUTINOVIC,
AND FAHRENHOLZ
._^ .
. ..^
_.
-. 1, -.
-_--
-
FIG. 5. Amino acid analysis diagram of [3H]secretin. Conditions are the same as those given in the Legend to Fig. 2. Column temperature is 66°C.
and [3H]secretin are 27% (K,:3.7 x lop8 M) and 56% (K,: 1.8 x 1Om8M) of unmodified secretin (K,: 1.Ol x lo+’ M), respectively. Secretin, treated as described for iodination but in the absence of ICI, gives the same K, value as untreated secretin. A large excess of ICl used for iodination strongly reduces the apparent affinity (and also the potency) of iodinated derivatives in stimulating the enzyme (Table 1). At the concentration of 6 x 10e8 M, [3H]secretin binds rapidly to pancreatic plasma membranes, attainig a constant level of bound hormone within 1 min, the earliest instant at which measurements can be performed. This constant level of bound hormone is maintained for 40 min (Fig. 7). Adding excess unlabeled secretin reduces the tracer binding by about 70%, suggesting that the same sites are involved in the binding of unlabeled secretin. DISCUSSION
We have demonstrated that it is possible to iodinate the N-terminal histidyl residue of secretin, leading to an almost 100% yield of mono- and
SECRETIN
PREPARATION
Secret!n
595
AND ISOLATION
[M]
FIG. 6. Biological activity of iodinated secretin and tritiated secretin as assayed by the ability of these peptides to stimulate adenylate cyclase in pancreatic plasma membranes. The incubation procedure is described under Materials and Methods. Only the peptidestimulated activity is shown. Each point represents a mean of two measurements: synthetic secretin (X). iodinated secretin (A), and tritiated secretin (0).
disubstituted derivatives and also to replace all of the iodine atoms against tritium and hydrogen by a catalytic exchange reaction. The decreased affinity of iodosecretin toward pancreatic adenylcyclase cannot be attributed to the reaction conditions. In fact, it results from a substitution of histidine by one or two large iodine atoms, which lower the pKa for the dissociation of the imidazolium ion (19). A large excess of ICI apparently leads to undesirable side reactions, presumably by oxidation of the imidazole ring (25). This has been observed for histidine and histidine dipeptides by other workers (6,26). As expected, [3H]secretin exhibits a higher affinity for pancreatic adenylcyclase than iodosecretin but a lower affinity relative to unmodified secretin. Such a decrease in biological activity of E3H]secretin may be explained by the instability of secretin in solution during tritiation (27). The rather low specific activity [3H]secretin shows that the exchange of TABLE THE
DEPENDENCE
OF THE
APPARENT
OF ICISECRETIN
AFFINITY USED
I OF IODINATED
SECRETIN
ON THE
FOR IODINATION
1CI:secretin
Apparent affinity (%)
0: 1 (control) 2.5:l 4:l IO:1
100 38 25-30 14
RATIO
596
LIN, MILUTINOVIC,
2wo
f‘* i
0
AND FAHRENHOLZ
.
.
10
20 Time
.
30
I.0
[mln]
FLG. 7. Time course of binding of [3H]secretin to pancreatic plasma membranes. [3H]secretin (6 x lo-I0 M) was incubated with plasma membranes (0.25 mgiml) at 22°C both in the presence (0) and absence (0) of unlabeled synthetic secretin (I x 1Om6M) in the incubation mixture containing 20 mM Tris-Cl (pH 7.4), 1% albumin, 1 mg/ml soybean trypsin inhibitor, and 0.5 mM phenyl methyl sulfonyl fluoride (PMSF). The binding was determined at the indicated times on 400~~1 aliquots, as described under Materials and Methods. Each point represents the mean of three measurements.
halogen against tritium is accompanied by a tritium/hydrogen exchange. The small part of radioactivity which is located in the phenylalanine residue is probably due to labeling at the benzylic position (28). Since the specific activity of [3H]secretin is high enough for binding experiments and does not change during incubation, it is a useful tool for studying the interaction between the hormone and its receptor. ACKNOWLEDGMENTS The authors wish to thank Dr. E. Wiinsch for the supply of synthetic secretin and for his constructive suggetions. We thank Dr. I. Schulz for helpful discussions. We are grateful to Dr. E. Noppel for his assistance in the tritiation of iodo-secretin. The skillful technical assistance of W. Burkhardt, G. Schimmack. and K. Heil is also gratefully acknowledged.
REFERENCES 1. Harper, A. A., and Raper, H. (1943)J. Physiol. 102, 115-125. 2. Bodanszky, M. (1977) in Hormonal Receptors in Digestive Tract Physiology (Boufils, S., Fromageot, P., and Rosselin, G., eds.), pp. 13-18, North-Holland, Amsterdam. 3. Wiinsch, E., Jaeger, E., Moroder, L., and Schulz, I. in Hormonal Receptors in Digestive Tract Physiology (Boufils, S., Fromageot, P., and Rosselin, G., eds.), pp. 19-27, North-Holland, Amsterdam. 4. Milutinovic, S., Schulz, I., and Rosselin, G. (1976)Biochim. Biophys. Acra 436, 113- 127. 5. Hunter, W. M., and Greenwood, F. C. (1962) Nature (LondonJ 194, 495-496. 6. Wolf, J., and Covelh, J. (1969) /. Biochem. 9, 371-377. 7. Tai, H. H., Korsch, B., and Chey, W. Y. (1975) Anal. Biochem. 69, 34-41.
SECRETIN
PREPARATION
AND ISOLATION
597
8. Schaffalitzkyde Muckadell, 0. B., and Fahrenkrug, J. (1976)Scnnd. J. C/in. Lab. Znvesr. 36, 661-667. 9. Holohan, K. N., Murphy, R. F., Flangan, R. W. J., Buchanan, K. D., and Elmore, D. T. (1973) Biochim. Biophys. Acra 322, 178-180. 10. Desbuquois, B. (1974) Eur. J. Biochem. 46, 439-450. 11. Ondetti, M. A.. Sheehan, I. T., and Bodanszky, M. (1968) in Pharmacology of Hormonal Polypeptides and Proteins (Back, N., Martini, L., and Paoletti, R., eds.), pp. 18. Plenum, New York. 12. Taylor, S. I. (1975) Biochemisrry 14, 2357-2361. 13. Morgat. J. L., Hung, L. T., and Fromageot, P. (1970) J. Lab. Compounds 6, 276-284. 14. Morgat, J. L., Girma. J. P.. and Fromageot, P. in Hormonal Receptors in Digestive Tract Physiology (Bout’ils, S., Fromageot. P.. and Rosselin, G., eds.). pp. 43-53, NorthHolland, Amsterdam. 15. Ahlroth, A., and Mutt, V. (1970) Anal. Biochem. 37, 125-128. 16. Postmes, T. (1963) Acra Endocrinol. 42, 153-162. 17. Wiinsch. E. (1972) Naturwissenschaft 59, 239-246. 18. Wiinsch, E. Personal communication. 19. Brunigs, K. J. (1947)J. Amer. Chem. Sot. 69, 205-208. 20. Savoie, J. C., Thomopoulos, P.. and Savoie, F. (1973) J. C/in. Invest. 52, 106-114. 21. Holloway. C. T.. Bond, R. P. M., Knight, I. G., and R. B. Beechey. (1%7)Biochemistry 6, 19-24. 22. Krishna, G., Weiss, B., and Brodie, B. B. (1968) J. Pharmacol. Exp. Ther. 163,379-385. 23. Frankel-Conrat, H. (1950) Arch. Biochem. 27, 109-124. 24. Koshland, M. E.. Englberger, F. M., Erwin, F. M., and Gaddone, S. M. (1963) J. Biol. Chem. 238, 1343-1348. 25. Pinner, A., and Schwarz, R. (1902) Chem. Ber. 35, 2441-2459. 26. Glazer, A. N., and Sanger, F. (1964) Biochem. J. 90, 92-98. 27. Grossmann, M. I. (1969) Gastroenterology 57, 767. 28. Evans, E. A., Sheppard, H. C., Turner, J. C., and Warell, D. C. (1974) J. Lab. Compounds
6, 276-284.
BIOCHEMISTRY
Preparation
88, 587-597 (1978)
and Characterization of Biologically Active lodo- and C3H]Secretin
JIANN-TRZUO LIN, SLOBODAN MILUTINOVIC, AND FALKFAHRENHOLZ Max-Planck-Institut
ftir
Biopltysik,
FrankfurtlMain,
Germany
Received October 14, 1977; accepted March 17. 1978 Synthetic secretin has been iodinated at the N-terminal histidine, leading to an almost 100% yield of mono- and diiodo-secretin (“iodo-secretin”). The catalytic exchange of iodine against tritium results in the preparation of secretin labeled with tritium mainly at the hktidine residue (7 Ciimmol). Iodo-secretin and [YH]secretin have the same potency in stimulating pancreatic adenylate cyclase as secretin, but the apparent affinity of [3H]secretin for this enzyme is twice as high as for iodo-secretin. [3H]Secretin binds rapidly to pancreatic plasma membranes. Adding excess unlabeled secretin reduces the tracer binding by about 70%.
It is well known that the peptide hormone secretin regulates electrolyte and water secretion of the pancreas (1). Conclusions about the nature of its receptor have been deduced indirectly from structure activity studies by Bodanszky (2) and Wunsch et al. (3). The interaction of rz51-labeled secretin with pancreatic plasma membranes was studied by Milutinovic ef al. (4), and the results were interpreted to mean that secretin and 1251labeled secretin have high affinity to the same binding site. The labeled derivative, however, which was prepared with 1251Na and chloramine-T according to the method of Hunter and Greenwood (5), in contrast with secretin itself, did not stimulate pancreatic adenylate cyclase. Radioactive labeling of secretin is particularly difficult for want of tyrosine; besides, iodination at the N-terminal histidine is determined by the low reactivity of the imidazole nucleus (6). Introduction of iodine into secretin by the chloramine-T method (7,8) or the lactoperoxidase method (9) lead to a mixture of labeled and unlabeled secretin with high specific radioactivity used in radioimmunoassay studies. A higher yield of iodinated peptides but with a much lower specific radioactivity can be obtained by using iodine monochloride (ICl). There is evidence that incorporation of one atom of iodine per molecule of secretin by ICI causes a two- to threefold decrease in the apparent affinity toward pancreatic adenylcyclase (10). The introduction of the large iodine atom into the N-terminal histidine, which is indispensabie for full biological 587
0003-2697/78/0882-0587$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction m any form reserved.
588
LIN,
MILUTINOVIC,
AND
FAHRENHOLZ
activity of secretin (1 l), might also affect the affinity of the labeled hormone to its receptor. This, in turn, could lead to nonlinear Scatchard plots of binding data (12). These difficulties may be avoided by using tritiated secretin in binding experiments. Polypeptides can be labeled with tritium on the tyrosine or histidine by catalytic exchange reaction of iodine against tritium (13,14). This paper describes the preparation of biologically active [3H]secretin (labeled at the histidine residue via the iodo intermediate), suitable for a study of the interaction between the hormone and its receptor. MATERIALS
AND METHODS
Synthetic secretin acetate was a gift by Prof. E. Wiinsch, Max-Planck Institute of Biochemistry (Munich, Germany). The dipeptide histidylserine, acrylamide, N,N’-methylenebisacrylamide, and ammonium persulfate were purchased from Serva (Heidelberg, Germany); tritium gas, (a- 32P)labeled adenosine 5’-triphosphate (2-10 Ci/mmol, sodium salt), and [83H]adenosine-3’-5’-cyclic phosphate (27 Ci/mmol) were from Amersham Buchler (Braunschweig, Germany); soybean tripsin inhibitor, pyruvate kinase (crystalline suspension, 10 mg/ml), and phosphoenolpyruvate (trisodium salt) were from Boehringer (Mannheim, German); bacitracin and albumin fraction V were from Sigma; EHWPO filters (pore size, 0.5 pm) were from the Millipore Corp.; the catalyst 10% Pd/C and aminopeptidase M were from Carl Roth (Karlsruhe, Germany); Sephadex G-25 and Sephadex SP-C25 were from Phamacia Fine Chemicals (Sweden); 1251C1(50mCi/ mmol) was obtained from New England Nuclear (Boston, Massachusetts); ICI was from Merck-Schuchard (Darmstadt, Germany); and Whatman 3 MM paper for electrophoresis was from Whatman Biochemicals Ltd. (Maidston, England). The catalyst 10% Pd/C was activated at 120°C for 24 hr under vacuum. Unlabeled ICI was sublimed twice at 20°C under vacuum and stored in dry dioxane at 4°C. The ICI content was determined by titration with sodium thiosulfate. Amino acid analyses were performed with a system LC 6000 from Biotronik, Wissenschaftliche Gerlte GmbH, Germany. High voltage electrophoresis was carried out with the Pherograph Mini 65 from Hormuth-Vetter (Wiesloch, Germany); ultraviolet absorption spectra were recorded with the spectrophotometer, type DB-GT, from Beckman Instruments; radioactivity was measured with the autogammascintillation spectrometer, model 5100, from Packard, and the betascintillation spectrometer, model BF 5000, was from Friske & Horpfner GmbH (Karlsruhe, Germany). Column effluents were monitored with an Uvicord III from LKB (Sweden) at wavelengths of 205 and 256 nm. For monitoring at a wavelength of 220 nm, the spectrophotometer DB-GT was used with a flow cell of 300 ~1. Zodination of secretin. Secretin, 3 pmol, was dissolved in 0.5 ml of
SECRETIN
PREPARATION
AND ISOLATION
589
guanidine hydrochloride (7 M) and diluted with 2.5 ml of 0.2 M ammonium acetate/ammonia solution (PH 9.2), which had been cooled to 4°C. The solution was stirred vigorously in an ice bath, and 9 pmol of iodine monochloride containing lz51Cl as tracer (about lo6 cpm) in 80 ~1 of dry dioxane was added dropwise to the solution within 20 sec. After the solution was stirred for 1 min, the reaction was stopped by adding 90 ,ul of a 0.1 M aqueous hydrazinhydrate solution. All iodinated products were protected from light. Isolation and characterization of iodo-secretin. The reaction mixture was lyophilized, redissolved in 1 ml of 0.5% acetic acid, and applied to a Sephadex G-25 column (90 x 1.25 cm) equilibrated with 0.5% acetic acid at 4°C. Eluting was performed with the same medium, and in each fraction (1.8 ml) the radioactivity was measured. The radioactive fractions were lyophilized and examined by high-voltage paper electrophoresis at pH 6.2, by gel electrophoresis according to Ahlroth et al. (15), and by ultraviolet absorption spectroscopy. For detection of peptides on paper, we used the ninhydrin, Pauly, and FFCA’ (16) reagents. Acid hydrolyses for amino acid analysis were performed with 6 N HCl at 110°C for 20 hr in the presence of norleucine as standard. The yield of peptides was estimated by quantitative amino acid analysis of aliquots using glutamic acid or phenylalanine as a measure of the amount of peptides present. For enzymatic hydrolysis, 5 nmol of peptide, dissolved in 100 ~1 of 0.2 M triethylammonium acetate buffer (PH 8.0), was incubated at 37°C with 100 mU of aminopeptidase M dissolved in 50 ~1 of water. After 16 hr the pH was adjusted to be 3.5 by adding 1 N acetic acid and the mixture was evaporated to dryness. Tritiation. After iodination of 1.5 pmol of secretin we immediately added 0.5% acetic acid to the reaction mixture to adjust the pH to 8.3. The solution was kept frozen in liquid nitrogen until we transferred it into a special flask connected to a vacuum line similar to that described by Morgat et al. (13). The solution was frozen, and vacuum was applied to remove all gas which was dissolved. Pd/C catalyst (10% Pd) was then added, and 20 Ci of pure tritium gas was introduced at a pressure of about 500 mm Hg. The mixture was brought to 10°C and stirred magnetically. After an hour the solution was frozen again and tritium gas was removed. By adding 1 N acetic acid, we acidified and simultaneously diluted the mixture to a volume of 10 ml. Centrifugation, followed by repeated lyophilization, gave a white material which was redissolved in 0.5% acetic acid and stored in liquid nitrogen. Zsofation and purijication of [3H]secretin. To remove the rest of labile tritium and inorganic salts, the reaction mixture was filtered on a Sephadex G-25 column (90 x 1.25 cm), equilibrated with 0.5% acetic acid, and eluted 1 Ferric ferricyanide-orsenius
acid.
590
LIN,
MILUTINOVIC,
AND
FAHRENHOLZ
with the same solvent. To examine the eluant, the same procedure was used as for the identification of iodo-secretin. The fractions which contained P-radioactivity were collected and lyophilized. We dissolved the dry material in 0.01 M ammonium acetate buffer @H 6.0) and chromatographed it on a Sephadex SP-C25 column (5 x 0.7 cm) equilibrated with the same buffer. The chromatography was performed at 4”C, as described by Wtinsch et al. (17); ammonium acetate buffer, however, was used instead of ammonium carbonate buffer as the eluant (18). Fractions of 3.7 ml were collected. To determine the [3H]secretin-containing fractions, we applied the same analytical methods as those used for the characterization of iodo-secretin. To find the location of tritium label in the peptide chain, we digested rH]secretin by aminopeptidase M and applied the hydrolyzate to the amino acid analyser. Fractions of the eluant were collected, and the P-radioactivity in each was measured. Preparation of +iodo-L-histidine (MZH] and 2,4-diiodo-L-histidine (DZH). Iodo-histidines were prepared either according to the method of Brunings (19) or of Savoie et al. (20). The position of the iodine atom in
the imidazole ring was determined by nmr spectroscopy of the compounds dissolved in a mixture of DzO/CF3COOD (21). Stimulation of pancreatic adenylate cyclase activity. Pancreatic plasma membranes were prepared as described (4). Adenylate cyclase activity was measured essentially by the method of Krishna et al. (22) with some modifications (4). The following incubation medium was used: 0.8 mM [a32P]ATP, 5 mM MgC12, 40 mM Tris-Cl (PH 7.4), 10 mM theophylline, 80 to 120 pg of membrane protein, and an ATP-regenerating system containing 5 mM phosphoenlpyruvate and 0.2 mg of pyruvate kinase/ml. The final volume was 0.1 ml. Incubations were carried out at 30°C for 20 min and stopped by adding a 1% SDS solution. The apparent affinity of secretin derivatives was expressed as a percentage of the apparent affinity of secretin: (K, of secretin/K, of secretin derivative) x 100. Binding assay of secretin derivatives to pancreatic membranes. Membranes (150 pg of protein) were added to the preequilibrated assay mixture (described in legends to Fig. 7), and after incubation at 22°C for different times, the binding was determined by filtration through a Millipore filter, as described by Milutinovic et al. (4). RESULTS Zodosecretin. In a preliminary experiment we observed that a large excess of ICI or that long reaction times decreased the yield of iodinated dipeptide histidyl-serine (position l-2 of secretin). Therefore, iodination of secretin was carried out with 2.5 to 4 Eq of ICI at 4”C, the reaction being terminated after 1 min. The elution diagram (Fig. 1) of the reaction mixture on Sephadex G-25 shows two peaks of y-radioactivity, the first one
SECRETIN
PREPARATION
591
AND ISOLATION
k IW-
EO-
8 5 z 2 4
60-
LO-
20 -
O-
10
FIG. 1. SephadexG-25 column chromatography of iodinated secretin: 6) uv absorbance at 205 nm; t-a-e-0) uv absorbance at 254 nm; and (----) y-radioactivity. The first peak contains iodo-secretin.
(fractions 20-35) containing all eluted peptides and the second one (fractions S-80) containing iodide and salts. The degree to which secretin is modified can be examined by gel electrophoresis and amino acid analysis of the peptides eluted in the first peak. Only traces of secretin show up in gel electrophoresis studies. After digestion with aminopeptidase M, the amino acid analysis reveals the presence of MIH and DIH (MIH:DIH = 2: 1 for 2.5 Eq of ICI and =l:l for 4 Eq of ICl) instead of histidine. The exact ratio of iodohistidines is difficult to determine, since the hydrolysis of secretin, iodinated at its N-terminal residue with aminopeptidase M, is much slower than that of unmodified secretin. Also, conversion of some DIH to MIH during enzymatic digestion has been reported (20). After hydrolysis of iodinated secretin by 6 N HCl some histidine is found (Phe:His = 1:O.l; Fig. 2), mainly because of the complete destruction of DIH and the partial destruction of MIH (23, 24). From the preceding it is obvious that secretin is iodinated at its N-terminal histidyl residue with a nearly 100% yield, leading to mono- and disubstituted derivatives (iodosecretin). During gel electrophoresis in the system of Ahlroth and Mutt (15) and during high voltage paper electrophoresis at pH 6.5, iodo-secretin migrates toward the cathode more slowly than secretin. Iodo-secretin gives an intense blue color reaction with FFCA reagent and only gives a faint color with Pauly reagent and ninhydrin. Its ultraviolet absorption spectrum in 0.5% acetic acid shows an additional maximum at 274 nm (E = 2140). [3H]Secretin. Iodine can be completely exchanged against tritium and hydrogen by treating an aqueous solution of iodosecretin at pH 8.3 in the presence of 10% Pd/C. After the reaction mixture is filtered by Sephadex G-25, the eluted peptides seem to contain no y-radioactivity. Gel elec-
592
LIN, MILUTINOVIC,
AND FAHRENHOLZ
FIG. 2. Amino acid analysis diagram of iodinated secretin after hydrolysis with 6 N HCl at 11°C for 20 hr. Conditions: column, 45 x 0.6 cm; Durum resin DCdA: temperature, 70°C; eluting buffer, pH 6.35; and 1.4 N Na+.
trophoresis shows one major band corresponding to [3H]secretin and two minor components (Fig. 3); the latter can be removed by ion exchange chromatography with Sephadex SP-C25 (Fig. 4). Amino acid analysis of chemically pure tritiated secretin (fractions 99-153) gives a one-to-one ratio of histidine to phenylalanine (Fig. 5). We find that most of the pradioactivity is incorporated into histidine; however, 15% of the specific radioactivity of histidine is located in the phenylalanyl residue. The specific radioactivity of [3H]secretin is found to be 7 Cilmmol (theoretical: 3060Ci/mmol), and the yield of tritiated secretin is 15% of the initial unlabeled secretin. The results reported above were obtained by tritiation of iodosecretin in ammonium acetate buffer in the presence of an excess of iodide. When the iodide is removed and the reaction is performed in 0.2 M borate buffer @H 8.3), the results remain essentially unchanged.
SECRETIN
PREPARATION
593
AND ISOLATION
FIG. 3. Gel electrophoresis of rYH]secretin in the system described by Ahlroth et al. (15): (A) [3H]secretin before purification; (B) [3H]secretin after purification on Sephadex SP-C25 (fractions 99-153); (C) synthetic secretin; and (D) [3H]secretin plus secretin.
Tritiation of secretin treated for iodination with a large excess of ICI (10 equivalents) for 10 min yields a much lower specific radioactivity. The low ratio of histidine to phenylalanine (0.2: 1) obtained by amino acid analysis of this tritiated material indicates the occurrence of side reactions at the histidyl residue during iodination. Biological activity. Iodo-secretin and [3H]secretin stimulate pancreatic adenylcyclase to the same extent as secretin. The K, values computed graphically from Fig. 6 show that the apparent affinities of iodo-secretin b
105PH
T
C[Ml
E s
lOI-
ij s H 103-
I
102Fraction
0
numba
FIG. 4. Purification of [3H]secretin by chromatography on Sephadex SP-C25: (----) uv absorbance at 220 nm, (-) P-radioactivity, (-e-o-o) concentration of eluting buffer, and (- - -) pH of eluting buffer. Fractions 99 to 153 contain [3H]secretin.
594
LIN, MILUTINOVIC,
AND FAHRENHOLZ
._^ .
. ..^
_.
-. 1, -.
-_--
-
FIG. 5. Amino acid analysis diagram of [3H]secretin. Conditions are the same as those given in the Legend to Fig. 2. Column temperature is 66°C.
and [3H]secretin are 27% (K,:3.7 x lop8 M) and 56% (K,: 1.8 x 1Om8M) of unmodified secretin (K,: 1.Ol x lo+’ M), respectively. Secretin, treated as described for iodination but in the absence of ICI, gives the same K, value as untreated secretin. A large excess of ICl used for iodination strongly reduces the apparent affinity (and also the potency) of iodinated derivatives in stimulating the enzyme (Table 1). At the concentration of 6 x 10e8 M, [3H]secretin binds rapidly to pancreatic plasma membranes, attainig a constant level of bound hormone within 1 min, the earliest instant at which measurements can be performed. This constant level of bound hormone is maintained for 40 min (Fig. 7). Adding excess unlabeled secretin reduces the tracer binding by about 70%, suggesting that the same sites are involved in the binding of unlabeled secretin. DISCUSSION
We have demonstrated that it is possible to iodinate the N-terminal histidyl residue of secretin, leading to an almost 100% yield of mono- and
SECRETIN
PREPARATION
Secret!n
595
AND ISOLATION
[M]
FIG. 6. Biological activity of iodinated secretin and tritiated secretin as assayed by the ability of these peptides to stimulate adenylate cyclase in pancreatic plasma membranes. The incubation procedure is described under Materials and Methods. Only the peptidestimulated activity is shown. Each point represents a mean of two measurements: synthetic secretin (X). iodinated secretin (A), and tritiated secretin (0).
disubstituted derivatives and also to replace all of the iodine atoms against tritium and hydrogen by a catalytic exchange reaction. The decreased affinity of iodosecretin toward pancreatic adenylcyclase cannot be attributed to the reaction conditions. In fact, it results from a substitution of histidine by one or two large iodine atoms, which lower the pKa for the dissociation of the imidazolium ion (19). A large excess of ICI apparently leads to undesirable side reactions, presumably by oxidation of the imidazole ring (25). This has been observed for histidine and histidine dipeptides by other workers (6,26). As expected, [3H]secretin exhibits a higher affinity for pancreatic adenylcyclase than iodosecretin but a lower affinity relative to unmodified secretin. Such a decrease in biological activity of E3H]secretin may be explained by the instability of secretin in solution during tritiation (27). The rather low specific activity [3H]secretin shows that the exchange of TABLE THE
DEPENDENCE
OF THE
APPARENT
OF ICISECRETIN
AFFINITY USED
I OF IODINATED
SECRETIN
ON THE
FOR IODINATION
1CI:secretin
Apparent affinity (%)
0: 1 (control) 2.5:l 4:l IO:1
100 38 25-30 14
RATIO
596
LIN, MILUTINOVIC,
2wo
f‘* i
0
AND FAHRENHOLZ
.
.
10
20 Time
.
30
I.0
[mln]
FLG. 7. Time course of binding of [3H]secretin to pancreatic plasma membranes. [3H]secretin (6 x lo-I0 M) was incubated with plasma membranes (0.25 mgiml) at 22°C both in the presence (0) and absence (0) of unlabeled synthetic secretin (I x 1Om6M) in the incubation mixture containing 20 mM Tris-Cl (pH 7.4), 1% albumin, 1 mg/ml soybean trypsin inhibitor, and 0.5 mM phenyl methyl sulfonyl fluoride (PMSF). The binding was determined at the indicated times on 400~~1 aliquots, as described under Materials and Methods. Each point represents the mean of three measurements.
halogen against tritium is accompanied by a tritium/hydrogen exchange. The small part of radioactivity which is located in the phenylalanine residue is probably due to labeling at the benzylic position (28). Since the specific activity of [3H]secretin is high enough for binding experiments and does not change during incubation, it is a useful tool for studying the interaction between the hormone and its receptor. ACKNOWLEDGMENTS The authors wish to thank Dr. E. Wiinsch for the supply of synthetic secretin and for his constructive suggetions. We thank Dr. I. Schulz for helpful discussions. We are grateful to Dr. E. Noppel for his assistance in the tritiation of iodo-secretin. The skillful technical assistance of W. Burkhardt, G. Schimmack. and K. Heil is also gratefully acknowledged.
REFERENCES 1. Harper, A. A., and Raper, H. (1943)J. Physiol. 102, 115-125. 2. Bodanszky, M. (1977) in Hormonal Receptors in Digestive Tract Physiology (Boufils, S., Fromageot, P., and Rosselin, G., eds.), pp. 13-18, North-Holland, Amsterdam. 3. Wiinsch, E., Jaeger, E., Moroder, L., and Schulz, I. in Hormonal Receptors in Digestive Tract Physiology (Boufils, S., Fromageot, P., and Rosselin, G., eds.), pp. 19-27, North-Holland, Amsterdam. 4. Milutinovic, S., Schulz, I., and Rosselin, G. (1976)Biochim. Biophys. Acra 436, 113- 127. 5. Hunter, W. M., and Greenwood, F. C. (1962) Nature (LondonJ 194, 495-496. 6. Wolf, J., and Covelh, J. (1969) /. Biochem. 9, 371-377. 7. Tai, H. H., Korsch, B., and Chey, W. Y. (1975) Anal. Biochem. 69, 34-41.
SECRETIN
PREPARATION
AND ISOLATION
597
8. Schaffalitzkyde Muckadell, 0. B., and Fahrenkrug, J. (1976)Scnnd. J. C/in. Lab. Znvesr. 36, 661-667. 9. Holohan, K. N., Murphy, R. F., Flangan, R. W. J., Buchanan, K. D., and Elmore, D. T. (1973) Biochim. Biophys. Acra 322, 178-180. 10. Desbuquois, B. (1974) Eur. J. Biochem. 46, 439-450. 11. Ondetti, M. A.. Sheehan, I. T., and Bodanszky, M. (1968) in Pharmacology of Hormonal Polypeptides and Proteins (Back, N., Martini, L., and Paoletti, R., eds.), pp. 18. Plenum, New York. 12. Taylor, S. I. (1975) Biochemisrry 14, 2357-2361. 13. Morgat. J. L., Hung, L. T., and Fromageot, P. (1970) J. Lab. Compounds 6, 276-284. 14. Morgat, J. L., Girma. J. P.. and Fromageot, P. in Hormonal Receptors in Digestive Tract Physiology (Bout’ils, S., Fromageot. P.. and Rosselin, G., eds.). pp. 43-53, NorthHolland, Amsterdam. 15. Ahlroth, A., and Mutt, V. (1970) Anal. Biochem. 37, 125-128. 16. Postmes, T. (1963) Acra Endocrinol. 42, 153-162. 17. Wiinsch. E. (1972) Naturwissenschaft 59, 239-246. 18. Wiinsch, E. Personal communication. 19. Brunigs, K. J. (1947)J. Amer. Chem. Sot. 69, 205-208. 20. Savoie, J. C., Thomopoulos, P.. and Savoie, F. (1973) J. C/in. Invest. 52, 106-114. 21. Holloway. C. T.. Bond, R. P. M., Knight, I. G., and R. B. Beechey. (1%7)Biochemistry 6, 19-24. 22. Krishna, G., Weiss, B., and Brodie, B. B. (1968) J. Pharmacol. Exp. Ther. 163,379-385. 23. Frankel-Conrat, H. (1950) Arch. Biochem. 27, 109-124. 24. Koshland, M. E.. Englberger, F. M., Erwin, F. M., and Gaddone, S. M. (1963) J. Biol. Chem. 238, 1343-1348. 25. Pinner, A., and Schwarz, R. (1902) Chem. Ber. 35, 2441-2459. 26. Glazer, A. N., and Sanger, F. (1964) Biochem. J. 90, 92-98. 27. Grossmann, M. I. (1969) Gastroenterology 57, 767. 28. Evans, E. A., Sheppard, H. C., Turner, J. C., and Warell, D. C. (1974) J. Lab. Compounds
6, 276-284.
