Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11985-11989, December 1995 Biochemistry

Two alternative processing pathways for a preprohormone: A bioactive form of VALENTINA BONETTO, HANS JORNVALL, VIKTOR MUTT, AND RANNAR SILLARD* Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77, Stockholm, Sweden Contributed by Viktor Mutt, September 5, 1995

ABSTRACT An N-terminally 9-residue elongated form of a cloned cDNA species (10, 13). This discrepancy was ex- secretin, secretin-(-9 to 27) amide, was isolated from porcine plained in terms of alternative splicing and later the shorter intestinal tissue and characterized. Current knowledge about form of cDNA was also found (14). A proform of secretin processing sites does not allow unambiguous predic- elongated at its C terminus by Gly-Lys-Arg has been described tion of the signal peptide cleavage site in preprosecretin but (15), and it might represent the next product in the C-terminal suggests cleavage in the region ofresidues -10 to -14 counted processing, presumably produced by cleavage with an enzyme upstream from the N terminus of the hormone. However, the specific for a dibasic site. The next, shortened form of the structure of the isolated peptide suggests that the cleavage precursor, secretin extended at its C terminus by glycine, has between the signal peptide and the N-terminal propeptide also been isolated (16). This form is presumably obtained by occurs at the C-terminal side of residue -10. Moreover, the carboxypeptidase action on the Gly-Lys-Arg-extended form. isolated peptide demonstrates that secretin can be fully pro- The mature hormone-C-terminally amidated secretin-is cessed C-terminally prior to the final N-terminal cleavage. produced from the glycine-extended peptide by the combined The results from this report, and those from earlier studies, action of a peptidylglycine a-monooxygenase and a peptidyl- where C-terminally elongated variants were isolated, show amidoglycolate lyase (17). In aHl of these proforms, the N that the processing of the secretin precursor may proceed by terminus starts with secretin itself, and thus cleavage of the one oftwo alternative pathways, in which either ofthe two ends N-terminal flanking peptide has been considered to be the is processed first. The bioactivity of the N-terminally extended initial processing event after removal of the signal peptide (13). peptide on exocrine pancreatic was lower than that In our laboratory, a variant form of secretin in which the of secretin, indicating the importance of the finally processed N-terminal tryptic peptide differs from that of secretin was free N terminus of the hormone for interaction with secretin observed earlier but was not further characterized (18). receptors. The present study demonstrates that an alternative process- ing pathway is possible, where the N terminus remains ex- Secretin, a gastrointestinal hormone, was discovered by Bayliss tended throughout processing of the C terminus, starting with and Starling in 1902 (1), isolated (2), and characterized as a the precursor and ending with arnidation of the C-terminal 27-residue C-terminally amidated peptide (3). It is known to be valine. produced in endocrine S cells of the small intestine (4, 5), pancreatic 13 cells (6), and possibly also in brain (7, 8). Its best MATERIALS AND METHODS known function is stimulation of the secretion of bicarbonate- rich pancreatic fluid (9). Peptide Purification. A concentrate of thermostable intes- The structures of the rat and mouse secretin precursors as tinal , CTIP, was prepared from porcine intestines as deduced from the corresponding cDNA sequences contain a described (19-21). Briefly, the uppermost meter of the intes- signal sequence, an N-terminal flanking peptide, secretin, and tines was boiled for 10 min, frozen, minced, and extracted with a C-terminal extension peptide in that order (10, 11). The 0.5 M acetic acid (20 h at 4°C). Peptides in the extract were sequence of pig preprosecretin has also been so adsorbed onto alginic acid (pH 2.7 ± 0.1), eluted with 0.2 M deduced, but it is probably lacking a few residues at the N HCl, and precipitated with salt (pH 3.5 ± 0.1) to produce the terminus of the signal peptide (10). The amino acid sequence CTIP. An aqueous solution of CTIP was fractionated with of the precursor is such that current knowledge about signal ethanol as described (22), and the peptides soluble at -20°C peptide cleavage sites (12) does not allow unambiguous pre- were further processed by size-exclusion chromatography on a diction of this site. On the basis of the amino acid sequence, Sephadex G-25 column. A fraction of low molecular weight the cleavage of the signal peptide has been proposed to occur peptides was extracted with methanol, and the peptides, at the C-terminal side of any of the residues between -10 and methanol soluble at neutral pH, were applied to a CM-22 -14 (10), most likely after residue -12, -13, or -14. Hence, column (14 x 20 cm). From this chromatography step, three for determination of the actual cleavage site, isolation of the large fractions were collected: presecretin, a second fraction corresponding N-terminally elongated form of secretin is that contains mainly secretin, and a more basic fraction necessary. In this report, we show that cleavage of the signal denoted postsecretin normally used for isolation of vasoactive peptide probably occurs at the C-terminal side of residue -10, intestinal peptide (23). This material was then used for further thus suggesting the position of cleavage and the length of the purification by ion-exchange chromatography on carboxy- N-terminal flanking peptide. methyl cellulose (CM-22, Whatman; 5 x 18 cm) in 0.0125 M It has been shown earlier that other secretin proforms also sodium phosphate (pH 6.4). Peptides were eluted with a linear exist in intestinal tissue. A proform, consisting of the secretin gradient of 0-0.3 M NaCl in the same buffer until the part together with its C-terminal flanking peptide, has been absorbance at 280 nm decreased below 0.1 (24). The column isolated, although the length of the latter was found to be 31 was then washed with 0.2 M HCl. NaCl was removed from the residues shorter than the amino acid sequence deduced from eluted peptides by passing the eluate through a column of Sephadex G-25 coarse in 0.2 M acetic acid and lyophilizing the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviation: TFA, trifluoroacetic acid. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 11985 Downloaded by guest on September 28, 2021 11986 Biochemistry: Bonetto et al. Proc. Natl. Acad. Sci. USA 92 (1995)

peptide-containing fractions. The fractions eluting between 78 using an uncoated capillary of 75 ,um diameter essentially as and 92 min contained the secretin-(-9 to 27) amide (N- described (25). Two different buffer systems were used (50 mM prosecretin) and were taken for further purification as de- phosphate buffers at pH 2.5 and pH 7.0). scribed below. Structural Analysis. The purified peptide was degraded in Porcine secretin was isolated as described (2) and used as a an Applied Biosystems sequencer (model 470A) and the standard in structural and bioactivity measurements. fractions were analyzed on a Hewlett-Packard HPLC system Reverse-Phase HPLC. The peptide fractions from cation- HP1090. Amino acid compositions were determined with an exchange chromatography were applied to LKB Ultropac TSK LKB Alpha Plus 4151 analyzer. The molecular mass of the ODS (7.8 x 300 mm). The column was eluted with a gradient peptide was measured on a Finnigan-MAT (San Jose, CA) of acetonitrile in 0.1% aqueous trifluoroacetic acid (TFA) Lasermat 2000, using a-cyano-4-hydroxycinnamic acid as the using buffers A (0.1% TFA/water) and B (0.1% TFA/water/ matrix. An aliquot (0.5 ,ul) of the sample, dissolved in 0.1% 80% acetonitrile) with 25-55% buffer B in 40 min. For TFA/70% acetonitrile/water, and of the matrix-saturated cation-exchange HPLC, Vydac (Hesperia, CA) 400VHP552 solution (0.5 ,ul in the same solvent) were mixed on a stainless (5.0 x 25 mm) was used. A NaCl gradient was employed using steel target and allowed to dry at room temperature. Mass buffers A (0.1% TFA/water/25% acetonitrile) and B (1 M spectra were obtained from the average of 10 laser shot NaCl in buffer A) with 0-30% buffer B in 30 min. The peptide recordings at 337 nm. Bovine (Sigma) was used as an fraction from the ion-exchange HPLC step was then applied to internal standard. a Vydac C18 (4.6 x 250 mm) HPLC column and eluted with a Sequence searches were carried out with the FASTA program methanol gradient using buffers A (0.1% TFA/water) and B of the Genetics Computer Group software package (release (0.1% TFA/water/80% methanol) with 60-100% buffer B in 8.0) (26) in the Swiss-Prot (release 30.0) data base. 40 min. Synthetic Peptide. N-prosecretin was synthesized by Scha- Enzymatic Digestion. The isolated prosecretin, its synthetic fer-N (Copenhagen). The crude peptide was purified by re- replica, and secretin were each dissolved (1 mg/ml) in 0.1 M verse-phase HPLC on a Vydac C18 (4.6 x 250 mm) column ammonium bicarbonate (pH 7.9) containing 5% acetonitrile. using 0.1% TFA/water as buffer A and 0.1% TFA/water/80% Endoproteinase Glu-C (analytical grade; Boehringer Mann- acetonitrile as buffer B with a gradient of 35-48% buffer B in heim) was added at an enzyme/substrate ratio of 1:10. Diges- 26 min. The purified peptide was freeze-dried and analyzed by tion was carried out at 25°C for 8 h. capillary electrophoresis and mass spectrometry. Capillary Zone Electrophoresis. The procedure was done Bioactivity Measurements. The bioactivity of secretin and with a Beckman P/ACE 5510 capillary electrophoresis system the secretin precursor was measured on an anesthetized cat as

4 B

0.75- E C

c. a 0.5 - a) a.) 0 CM C ce .o 0.25- 0

0 5 lo 15 20 25 30 355

0.15 C') nnA (D C D 0 0., 4- 0.06-

0. 3- 0 0 C

C\0 0.04- 0)CD) 0.:2- O :.0 0 0.1 .0 0.02-

c) ______-I______I I I 0O ~ 15 20 25 30 35 40 5 6 7 8 9 10 Time (min) Time (min)

FIG. 1. Purification of porcine N-prosecretin. (A) Semipreparative reverse-phase HPLC. Column, Ultropac TSK ODS (7.8 x 300 mm); mobile phase, gradient of 25-55% in 40 min of 0.1% TFA/water/80% acetonitrile in 0.1% TFA/water; sample load, 3 mg. Fraction denoted by stippled area was subjected to further purification. (B) Ion-exchange HPLC. Column, Vydac 400VHP552 (5.0 x 25 mm); mobile phase, 0.1% TFA/water/25% acetonitrile (buffer A) and 1 M NaCl in buffer A (buffer B); gradient, 0-30% buffer B in 30 min. (C) Final purification of secretin precursor. Column, Vydac C18 (4.6 x 250 mm); mobile phase, 0.1% TFA/water (buffer A) and 0.1% TFA/water/80% methanol (buffer B); gradient, 60-100% buffer B in 40 min. Stippled area indicates purified peptide peak. (D) Capillary zone electrophoresis in 50 mM phosphate buffer at pH 2.5. Downloaded by guest on September 28, 2021 Biochemistry: Bonetto et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11987

described (27). Briefly, the peptides were dissolved in 0.9% purification steps, a pure peptide was obtained and its homo- NaCl containing L-cysteine (100 mg/liter). An aliquot was geneity was checked by capillary electrophoresis (Fig. 1). injected i.v. into the cat followed by 0.5 ml of the solvent, and Determination of its N-terminal amino acid sequence, and the pancreatic juice, secreted in response to the injected subsequently a search in the Swiss-Prot data base, showed an peptide, was collected and titrated. identical match with a part of the porcine secretin precursor sequence. The molecular mass of the peptide was found to be 4055.4 Da, which is in good agreement with the calculated RESULTS AND DISCUSSION value for N-terminally elongated and C-terminally amidated N-prosecretin was detected by sequence analysis of HPLC- secretin, N-prosecretin (4054.7 Da). However, the accuracy of purified peptides from a basic fraction of the ion-exchange the mass determination does not unambiguously define chromatographic step on CM-cellulose. After three HPLC whether the C terminus is amidated or not and the C-terminal nature was established by further analysis (see below). It is also 0.06 well known that the C terminus of amidated peptides is produced from a consensus sequence Gly-Lys-Arg, where the A amide is obtained from glycine in the extension (17). This is the sequence also in the secretin precursor where a C-terminal E valine becomes amidated. C-terminally incompletely pro- 0.04- 1 cessed secretins, secretin-Gly-Lys-Arg and secretin-Gly, have c,J been isolated (15, 16). To verify the structure of the isolated prosecretin we com- coC pared the capillary electrophoretic mobilities of the isolated -e002L peptide and its synthetic replica at pH 2.5, where the two 00 peptides comigrated (data not shown). To prove that the C-terminal parts of the isolated peptide and secretin have the same, amidated structure, capillary electrophoretic mobilities of fragments obtained by digestion of the isolated prosecretin and secretin were compared at pH 7.0 (Fig. 2). It is evident that 0.06 - after mixing of the two digests, the C-terminal fragments comigrate (peak 1 in Fig. 2), showing that the C-terminal fragment of the isolated prosecretin has the same charge as the corresponding fragment from secretin. The amino acid com- 000 position of the isolated secretin precursor is given in Table 1 and is in good agreement with its theoretical composition. 0 An amount of 210 gg of highly purified N-prosecretin was C obtained from 85 mg of the CM-cellulose chromatography fraction, which corresponds to 1200 kg of porcine intestinal tissue. However, we do not know the amounts of the peptide distributed between side fractions throughout the purification. For comparison, 16 mg of highly purified secretin is usually obtained from 1000 kg of intestine under optimal conditions and 450 Ag of C-terminally elongated variant of secretin was 0 2.5 5 7. 10 obtained from 17,500 kg of intestinal tissue (13). Thus, the 0.06- N-prosecretin yield is higher than that for the C-terminally extended form by a factor of 10. From the structures of the porcine, rat, and mouse secretin precursors deduced from the corresponding cDNAs, it has 0.04 -3 been suggested that cleavage of the signal peptide in porcine -e~ ~ ~ ~Tm0(m;n) secretin might occur at the C-terminal side of any of residues c'J -10 to -14 (10). The amino acid sequence of the secretin precursor does not allow a further, unambiguous prediction of 2 the signal peptide cleavage site. However, in the present study, FIG.~~~~~. 2. Deemnto ifteCtrinlsrcueofN 0.02- we find that the signal peptide has been cleaved after residue -10, thus suggesting the actual position of cleavage. 01 Table 1. Amino acid composition of identified secretin precursor Ala 2.9 (3) 2.5 75 1 0 Arg 7.0 (7) T'ime (min) Asp 2.1 (2) Glx 3.0 (3)

FIG. 2. Determination of the C-terminal structure of N- Gly 2.2 (2) prosecretin by capillary zone electrophoresis. (A) Cleavage products of His 1.0 (1) the isolated prosecretin. (B) Cleavage products of secretin. (C) Leu 6.0 (6) Mixture (1:1 ratio) of digests obtained inA and B. Isolated prosecretin Phe 0.8 (1) and secretin were cleaved with endoprotease Glu-C and digests were Pro 3.0 (4) analyzed by capillary zone electrophoresis in 50 mM phosphate buffer Ser 3.4 (4) (pH 7.0). Samples were injected for 5 sec by pressurization. Peak 1 Thr 1.9 (2) corresponds to the C-terminal proteolytic fragment of the isolated Val 1.1 (1) prosecretin or of secretin; peak 2 corresponds to the N-terminal proteolytic fragment of the isolated prosecretin; peak 3 corresponds to Values are molar ratios after acid hydrolysis; numbers in parenthe- the N-terminal proteolytic fragment of secretin. ses give values from the sum of the sequence assignment. Downloaded by guest on September 28, 2021 11988 Biochemistry: Bonetto et al. Proc. Natl. Acad. Sci. USA 92 (1995)

Signalpeptide Flank Secretin Porcine RALLLLLLLPPLLLLAGCAARPAPPRAPRHSDGTFTSELSRLRDSARLQRLLQGLV... Mouse MEPPLPTPMLLLLLLLLSSSAALPAPPRTPRHSDGMFTSELSRLQDSARLQRLLQGLV... Rat MEPLLPTPPLLLLLLLLSSSFVLPAPPRTPRHSDGTFTSELSRLQDSARLQRLLQGLV... FIG. 3. Alignment of porcine, rat, and mouse secretin precursor sequences. Signal peptide, N-terminal flanking peptide, and mature hormone are indicated as found in the porcine form. The amino acid sequences of secretin precursors from other cessing occurs, leading to several C-terminally elongated in- species are different from that of the porcine form (Fig. 3). termediate forms of secretin. According to the alternative However, the N-terminal flanking peptides are highly con- pathway (pathway I) now found, the is served, particularly the segments containing proline and ar- C-terminally fully processed prior to N-terminal processing. ginine, which are identical in the three species, suggesting that After signal peptide cleavage, the C-terminal flanking peptide charge and conformational characteristics of this part of the and the Lys-Arg residues are then eliminated from the C molecule are important. terminus, followed by amidation via the action of amidating The bioactivity of the peptide on pancreatic exocrine secre- enzymes on the glycine residue (17). An indication of such a tion was determined in an anesthetized cat (Table 2). The processing pathway also exists in the case of , where we results show that the N-terminally elongated form is bioactive have detected in brain an N-terminally 9-residue elongated but at a lower level than secretin itself. The precursor causes form (25). However, in that case, no evidence for an alternative a response similar to that of secretin but at "30 times higher processing pathway via C-terminally elongated galanin pre- doses. We also noticed that while the onset time of the cursors exists at present. However, such a pathway has been response by secretin is 1 min, that with the precursor is recently described for (CCK). In its processing longer, -3 min. scheme, an N-terminal flanking peptide remains attached to Interestingly, other secretin precursors with a free N termi- that hormone formation of the C-terminal amide nus and C-terminal elongations were found to have bioactiv- during (30). ities comparable to or even higher than that of secretin (13, 15), Thereafter, CCK-58 is formed from CCK-83. Thus, processing while the precursor form isolated in this study has a lower of the C terminus prior to N-terminal cleavage seems to be a bioactivity. The reasons for such activity profile differences are general pathway for processing of several hormones. However, not clear. However, it has been demonstrated that the C- here we demonstrate that both pathways may be important at terminal part of secretin is involved in stimulation of pancre- the same time. atic islet cAMP accumulation and potentiation of the glucose- In conclusion, the present study indicates that two alterna- stimulated insulin release (28), while the N-terminal part of tive pathways may lead to the same peptide product. secretin had no effect on cAMP production or insulin release. It has also been shown that deletion of the histidine residue at This work was supported by the Swedish Medical Research Council, to Pharmacia Research Foundation, Novo Nordisk Foundation, the the first position leads almost complete loss of activity on Royal Swedish Academy of Sciences, the National Board for Labo- pancreatic exocrine secretion (29). On the other hand, the ratory Animals, the Swedish Cancer Society, Karolinska Institutet, and present study indicates that N-terminal elongation also leads to a Fellowship from the University of Padua (V.B.). a decrease of activity. These observations indicate that the free N terminus of the peptide is essential, although it is not alone sufficient, for bioactivity. K R E'P'T'i The purified secretin precursor demonstrates that removal of the N-terminal flanking peptide is not necessarily the first processing event, in contrast to previous beliefs (13). Instead, GIK RL-.E'R'M'I'N'A'L...P'E'PT'I'l E the processing may proceed by one of two alternative pathways (Fig. 4). By the previously described pathway (pathway II), the 'I \ II signal peptide is cleaved from the prepropeptide, then the N-terminal flanking peptide, and finally the C-terminal pro- WFLANK g | SECRETIN 11 GJK R RETINN GICRMINALPE Table 2. Bioactivities of isolated N-prosecretin, its synthetic and secretin analog, FAK SECREI GA JKR Injection, Response, Injected peptide pmol ,umol of alkali Secretin 40 78 WFLANK SECRETINN* M4SECRETINA 3GI Secretin 40 74 Isolated N-prosecretin 1200 80 Isolated N-prosecretin 2400 160 Isolated N-prosecretin 2400 140 SECRET~ * Isolated N-prosecretin 1200 64 Secretin 80 126 FIG. 4. Two alternative processing pathways for preprosecretin. In common for both pathways, the signal peptide (Signal) is cleaved off. Secretin 40 78 Pathway I: The precursor is C-terminally fully processed before Synthetic N-prosecretin 2400 152 cleavage of the N-terminal flanking peptide (Flank). Here only the Synthetic N-prosecretin 2400 165 N-terminally elongated and C-terminally amidated form has so far Synthetic N-prosecretin 1200 62 been isolated (this study). Other N-terminally elongated forms are Secretin 80 120 hypothetical. Asterisks indicate the C-terminal amide structure. G, K, Secretin 40 60 and R are one-letter codes for amino acids. Pathway II: N-terminal Synthetic N-prosecretin 2400 115 processing precedes cleavage of the C-terminal flanking peptide Isolated N-prosecretin 2400 135 (C-terminal peptide), cleavage of arginine and lysine, and formation of C-terminal amide from glycine. All the intermediate forms in this Secretin 40 62 pathway have been isolated. Downloaded by guest on September 28, 2021 Biochemistry: Bonetto et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11989 1. Bayliss, W. M. & Starling, E. M. (1902) J. Physiol. (London) 28, 16. Carlquist, M. & Rokaeus, A (1984)J. Chromatogr. 296, 143-151. 325-353. 17. Katopodis, A. G., Ping, D., Smith, C. E. & May, S. W. (1991) 2. Jorpes, E., Mutt, V., Magnusson, S. & Steele, B. B. (1962) Biochemistry 30, 6189-6194. Biochem. Biophys. Res. Commun. 9, 275-279. 18. Tatemoto, K. (1980) in Gastrointestinal Hormones, ed. Glass, 3. Mutt, V., Jorpes, J. E. & Magnusson, S. (1970) Eur. J. Biochem. G. B. J. (Raven, New York), pp. 976-977. 15, 513-519. 19. Mutt, V. (1959) Ark. Kemi 15, 69-74. 4. Polak, J. M., Coulling, I., Bloom, S. & Pearse, A. G. E. (1971) 20. Mutt, V. (1978) in Gut Hormones, ed. Bloom, S. R. (Churchill Scand. J. Gastroenterol. 6, 739-744. Livingstone, Edinburgh), pp. 21-27. 5. Polak, J. M., Bloom, S., Coulling, I. & Pearse, A. G. E. (1971) Gut 21. Chen, Z.-w., Agerberth, B., Gell, K., Andersson, M., Mutt, V., 12, 605-610. Ostenson, C.-G., Efendic, S., Barros-Soderling, J., Persson, B. & 6. Wheeler, M. B., Nishitani, J., Buchan, A. M., Kopin, A. S., Chey, Jornvall, H. (1988) Eur. J. Biochem. 174, 239-245. W. Y., Chang, T. M. & Leiter, A. B. (1992) Mol. Cell. Biol. 12, 22. Lee, J.-Y., Boman, A., Chuanxin, S., Andersson, M., Jornvall, H., 3531-3539. Mutt, V. & Boman, H. G. (1989) Proc. Natl. Acad. Sci. USA 86, 7. Mutt, V., Carlquist, M. & Tatemoto, K. (1979) Life Sci. 25, 1703-1708. 9159-9162. 8. Itoh, N., Furuya, T., Ozaki, K., Ohta, M. & Kawasaki, T. (1991) 23. Said, S. I. & Mutt, V. (1972) Eur. J. Biochem. 28, 199-204. J. Biol. Chem. 266, 12595-12598. 24. Sillard, R., Jornvall, H. & Mutt, V. (1993) Biochem. Biophys. Res. 9. Mutt, V. (1988) in Advances in Metabolic Disorders, ed. Mutt, V. Commun. 195, 746-750. (Academic, San Diego), pp. 251-320. 25. Sillard, R., Rokaeus, A, Xu, Y., Carlquist, M., Bergman, T., 10. Kopin, A. S., Wheeler, M. B. & Leiter, A. B. (1990) Proc. Natl. Jornvall, H. & Mutt, V. (1992) Peptides 13, 1055-1060. Acad. Sci. USA 87, 2299-2303. 26. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids 11. Lan, M. S., Kajiyama, W., Donadel, G., Lu, J. & Notkins, A. L. Res. 12, 387-395. (1994) Biochem. Biophys. Res. Commun. 200, 1066-1071. 27. Mutt, V. & Soderberg, U. (1959) Ark. Kemi 15, 63-68. 12. von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690. 28. Kofod, H., Thams, P. & Holst, J. J. (1991) Int. J. Pept. Res. 13. Gafvelin, G., Jornvall, H. & Mutt, V. (1990) Proc. Natl. Acad. Sci. 37, 134-139. USA 87, 6781-6785. 29. Bodanszky, M. (1974) in Endocrinology of the Gut, eds. Chey, 14. Kopin, A. S., Wheeler, M. B., Nishitani, J., McBride, E. W., W. Y. & Brooks, F. P. (C. B. Slack, Thorofare, NJ), pp. 3-13. Chang, T. M., Chey, W. Y. & Leiter, A. B. (1991) Proc. Natl. 30. Eberlein, G. A., Eysselein, V. E., Davis, M. T., Lee, T. D., Acad. Sci. USA 88, 5335-5339. Shively, J. E., Grandt, D., Niebel, W., Williams, R., Moessner, J., 15. Gafvelin, G., Carlquist, M. & Mutt, V. (1985) FEBS Lett. 184, Zeeh, J., Meyer, E. H., Goebell, H. & Reeve, J. R., Jr. (1992) J. 347-352. Biol. Chem. 267, 1517-1521. Downloaded by guest on September 28, 2021