Interaction of gastrin with transferrin: Effects of ferric ions

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 263, No. 2, June, pp. 410-417, 1988

Interaction SANDRO *Melbourne

of Gastrin with Transferrin:

C. LONGANO, Tumour

JAROMIR GRAHAM

Effects of Ferric Ions

KNESEL,* GEOFFREY S. BALDWIN*tl

J. HOWLETT,

Biology Branch, Ludwig Institute for Cancer Research, P.O. Royal Victoria, 3050 Australia, and Russell Grimwade School of Biochemistry, University of Melbourne, Parkv&, Victoria, 3052 Australia Received

December

Melbuurne

AND

Hospital,

‘7.1987

The sedimentation behavior of ‘261-labeled gastrin has been studied as a function of Fe3+ ion concentration and pH. Both sedimentation velocity and sedimentation equilibrium experiments indicated that high-molecular-weight Fe3+-gastrin complexes were formed at pH 5.0 and pH 7.4. Self-association of gastrin alone was observed at pH values below 5.0. 1251-labeledgastrin bound to human serum apotransferrin at pH 7.4. Scatchard analysis of the gastrin-apotransferrin complex gave a Kd of approximately 6.4 pM at 37”C, with two binding sites per molecule of apotransferrin. No significant binding of gastrin to diferric transferrin was observed under the same conditions. The binding of gastrin to apotransferrin was inhibited by NaCl. The results are consistent with the hypothesis that gastrin and transferrin act synergistically in the uptake of dietary iron by the gastrointestinal tract. o 1988 Academic POW, IX

Human transferrin, the major iron binding protein in serum, consists of two homologous domains each possessing an independent metal binding site (1, 2). Transferrin is synthesized predominantly by the liver but also by testis, brain, bone marrow, submaxillary gland, spleen, and oviduct (3,4). Serum transferrin levels are increased by nutritional iron deficiency and by changes in hormone levels (5). Synthesis of testicular transferrin can also be affected by a number of hormones, including retinol, testosterone, insulin, and follicle-stimulating hormone (6). Recent studies have indicated that transferrin may mediate the uptake of iron from the intestine (7). Transferrin has been isolated from rat intestine (8), where it is located both within, and on the luminal surface of, the mucosal cells of the duodenum and jejunum (9). However, no 1 To whom 0003-9861188

correspondence

should

$3.00

Copyright Q 1988 by Academic Press. Inc. All righta of reproduction in any form reserved.

be addressed, 410

transferrin mRNA has been detected in the small intestine of either normal or iron-deficient rats (10). Absorption of both 5gFeand ‘%I-labeled transferrins has been observed from the lumen into mucosal cells of duodenal and jejunal segments of rat intestine (11). The following model has been proposed to explain these observations (10). Apotransferrin is synthesized in the liver, enters the intestine via the bile, and binds iron in the intestinal lumen. The loaded transferrin is taken up into the mucosal cells, where the iron is either deposited in mucosal ferritin, or released into the blood. Apotransferrin then returns to the brush border to be recycled. The increased levels of transferrin in bile and intestinal mucosa of iron-deficient rats are consistent with the model (10). The isolation of a variant transferrin from porcine gastric mucosa (12) suggests that in other species some of the intestinal transferrin may also originate from the stomach.

BINDING

OF GASTRIN

The hormone gastrin has long been recognized as a stimulant of acid secretion by the parietal cells of the gastric mucosa (13). An interaction between gastrin and porcine gastric and serum transferrins has recently been detected in vitro by covalent crosslinking (12). A gastrin concentration of approximately 100 VM was required to inhibit by 50% the crosslinking of ‘251-labeled gastrin to transferrin. Since significant amounts of gastrin are released into the gastric lumen in many species (14, 15) and since gastrin is known to bind metal ions (16, 17), we were interested in testing the hypothesis (12) that interaction between gastrin, ferric ions, and transferrin might be physiologically relevant. Thus gastrin might facilitate the delivery of dietary iron to apotransferrin by a mechanism analogous to the in vitro catalysis of transferrin loading by anionic chelating agents such as nitrilotriacetic acid (18). We have therefore investigated the interaction between gastrin and ferric ions at neutral and low pH in an ultracentrifuge. We have also analyzed the binding of gastrin to both apo- and diferrictransferrin by the same technique. EX.PERIMENTAL

MATERIALS

Human [Nlei5]-gastrin17 was from Research Plus (Bayonne, NJ). Gastrini, was iodinated and the monoiodinated component (sp act 950 pCi/ug) was purified by reverse-phase high-performance liquid chromatography as described by Baldwin and coworkers (19). Nalz51 (sp act 15.9 mCi/hg) was obtained from the Radiochemical Centre (Amersham, England). Human serum transferrin, obtained from Sigma (St. Louis, MO), migrated as a single band when electrophoresed on polyacrylamide gels, and contained <4% ferric transferrin, measured as its residual absorption at 470 nm. Bovine serum albumin (BSA)* and ovalbumin were from Sigma; ferric chloride from BDH (Kilsyth, Australia); Hepes (free acid) from Calbiochem-Behring (La Jolla, CA); Triton X-100 from Ajax (Cheltenham, Australia); Dextran T40 from Pharmacia (Uppsala, Sweden); and desferrioximine from Ciba-Geigy (Lane Cove, Australia).

* Abbreviations used: BSA, bovine serum albumin; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

411

TO TRANSFERRIN METHODS

Preparation of apotransferrin and diferrictransferrin. Apotransferrin was prepared by dialysis of human serum transferrin against 10 lllM Na+ acetate, pH 5.0, for at least 8 h with one or two buffer changes, followed by dialysis against 100 ml of the same buffer containing 50 pg/ml of desferrioximine to chelate any Fe3+ ions. The transferrin was reequilibrated to pH 7.4 by dialysis against 10 mM Na+ Hepes buffer. A stock solution of diferrictransferrin was prepared from apotransferrin by adding 2 eq of a solution containing 10 mM FeCb and 10 mM nitrilotriacetic acid in 1 mM Na+ Hepes, pH 7.4. The solution was mixed, left on ice for 30 min, and excess Fe-III-nitrilotriacetate was removed by extensive dialysis against 10 mM Na+ Hepes, pH 7.4. Diferrictransferrin prepared by this method had a visible absorption spectrum identical to that reported by Bates and Schlabach (18). Sedimentation velocity studies. The effect of iron, pH, or transferrin on the centrifugation behavior of ‘%I-labeled gastrin was determined using the TL-100 ultracentrifuge (Beckman). Samples of 100 ~1 were prepared in buffers containing either 5 mg/ml ovalbumin or Dextran T40 for density stabilization and centrifuged at 100,000 rpm for 55 min. At the conclusion of centrifugation the contents of the tube were fractionated into lo-111 aliquots using a microfractionator (Beckman) and the concentration of radiolabeled gastrin near the meniscus (top two lo-p1 aliquots) was determined and expressed as a percentage of the initial concentration. Sedimentation equilibrium studies. Sedimentation equilibrium distributions were formed by centrifugation of the sample (100 ~1) for 24 h in an air-driven ultracentrifuge (Beckman) as described previously (20) or for 48 h in a TL-100 ultracentrifuge. Ovalbumin (5 mg/ml) was included in all samples to provide density stabilization. Data obtained from the air-driven ultracentrifuge in the form of concentration (C) versus aliquot number were converted to plots of In C/C,, versus r2 where r is the radial distance in the centrifuge tube and Co is the initial concentration.

412

LONGANO

The relationship between the volume of each aliquot and the radial position in the ultracentrifuge tube has been described for the air-driven ultracentrifuge (20, 21). For the TL-100 ultracentrifuge the relationship has recently been considered (21) and is reproduced below with corrections. The volume (V) at radial positions r is given by V(r) = J

PI

A(r)dr

rnt

where A(r) is the cross-sectional area of the solution perpendicular to the radial path. For calculation of A(r) the tube is considered as a half sphere of radius a, joined to a cylinder. The radial positions with respect to the center of rotation r, and rb represent the inner and outer positions of the junction between the sphere and the cylinder and 8 is the angle of inclination of the tube to the vertical. With this formulation the value of A(r) becomes: For r d r,, A(r) = mz2 csc 8. For r, < r < r, + 2a cos 8

ET

AL.

specific volume; w is angular velocity; p is solution density; and T is absolute temperature. V and M for the ?-labeled gastrin were calculated from the amino acid composition to be 0.70 ml/g and 2205 respectively (22, 23). RESULTS

The Efect of Iron on the Sedimentation Behavior of Gust& The results in Table I show the effects of added iron on the sedimentation properties of ‘%I-labeled human [Nle”]-gastrin17. At pH 5.0 and in the absence of added iron the fraction of radiolabeled gastrin near the meniscus was approximately 70%. This fraction decreased as the concentration of added iron increased. At a concentration of 13.0 PM added iron approximately 95% of the radiolabeled gastrin was depleted from the meniscus. Results were also obtained for radiolabeled gastrin samples prepared in 10 mM Hepes buffer, pH 7.4, containing 5 mg/ml Dextran T40 and 0.002% Triton X-100 (Table I). In the absence of added iron the fracTABLE

A(r) = a2

5 -k LYm

+ arcsin ff

EFFECT

[( X (csc 8) + t - p m-

For r > r, + 2a cos 9

where r - r, set 8 a

and a sin 8 P=v 1 - (Y2cos2 8 For an ideal solute the slope of the In C/C, vs r2 plot is given by d In C/C,, = M(l - Vp)w2 dr2 2RT where M is molecular

‘l-gastrin meniscusb Added

Fe&” (PM)

0.0 0.1 1.3 13.0 130 1300

A(r) = ?ra2(1 - CX~COS%~),

a=l--

OFIRONONTHE SEDIMENTATIONBEHAVIOR OF'~I-LABELED GASTRIN

arcsin p w x (1 - cu%os28) . I

[31

weight; V is partial

I

at (%)

pH 5.0

pH 7.4

70.3 f 5.0 62.5 k 6.0 16.0 + 9.8 5.0 f. 2.3 3.5 f 1.1 3.9 * 1.0

80.0 * 1.5 80.7 2 0.7 79.1 53.5 5.8 3.5

k 0.9 t- 13.4 + 2.6 + 0.2

Note. Samples were sedimented in 10 mM Na+ acetate, pH 5.0, or 10 mM Na+ Hepes, pH 7.4. The samples also contained 5 mg/ml Dextran T40 and 0.002% Triton X-100. Centrifugation was at 100,000 rpm and 10°C for 55 min. a FeCla was added to give the final concentration of total iron as shown. * The values represent the fraction of ‘%I-labeled gastrim in the top two lo-p1 fractions of the centrifuge tube at the conclusion of centrifugation. Values are the average of three samples k 1 SD.

BINDING

OF GASTRIN

tion of radiolabeled gastrin at the meniscus was 80% of original while in the presence of 13.0 I.LM iron the fraction was 54% of original. Although the added iron under these conditions is less soluble (24) the results indicated that iron significantly increases the rate of sedimentation of radiolabeled gastrin at both pH 5.0 and pH 7.4. Complete sedimentation equilibrium profiles of lz51-labeled human [Nle15]-gastrim, at pH 7.4 in the presence and absence of iron are presented in Fig. 1. The data obtained in the absence of iron indicate no significant sedimentation of the gastrin under the conditions used. The line drawn through these data in Fig. 1 has been calculated for a species of molecular weight 2205 and a partial specific volume of 0.70 ml/g. The In C/C,, versus r2 plot for the data obtained in the presence of 1.3 mM FeC& (Fig. 1) indicates significant association of gastrin. Thus the concentration of radiolabeled gastrin at the meniscus at equilibrium is only 3.1% of the initial concentration. The concentration distribution at the bottom of the centri-

2,

1'

413

TO TRANSFERRIN

P"

FIG. 2. The effect of pH on the sedimentation of ‘?-labeled gastrin: Samples of ?-labeled gastrin (11,000 cpm, 3.5 fmol) were sedimented in buffers containing 10 mM Na+ citrate, 10 mM Na+ Hepes at different pH values (2.5-8.5) with 5 mg/ml Dextran T40 and 0.002% Triton X-100, using a TL-100 ultracentrifuge at 100,000 rpm and 1O’C for 55 min. Data are expressed as percentages of lzI-labeled gastrin remaining at the meniscus as a function of pH.

fuge tube (radial positions r = 1.2 cm to r = 1.4 cm) indicates the formation of higher-molecular-weight complexes. The slope of the best fit line drawn through the data obtained from this region yields a value of 114,700 for the weight-average molecular weight.

I, s 5

0.

-1

FIG. 1. The effect of ferric ions on the sedimentation of ‘l-labeled gastrin: ‘%I-labeled gastrin (11,000 cpm, 3.5 fmol) was sedimented to equilibrium in 10 mM Na+ Hepes, pH 7.4, 5 mg/ml ovalbumin in the absence (A) and in the presence (0) of 1.33 mM added Fe’+. Centrifugation was carried out in an Airfuge at 33,000 rpm and 10°C for 24 h.

The Efect of pH on the Association Gas&in

of

‘&I-labeled gastrin was sedimented at 100,000 rpm for 55 min in solutions of different pH ranging from 2.5 to 8.5. At pH 8.5, 78% of the radiolabeled gastrin remained near the meniscus (Fig. 2). Greater than 80% of the radiolabeled gastrin was depleted from the meniscus at pH 2.5. The midpoint of the titration curve for the sedimentation of radiolabeled gastrin occurred at approximately pH 4.5. A complete sedimentation equilibrium profile of iz51-labeled gastrin at pH 2.5 is presented in Fig. 3. The best fit line drawn through the data yields a weight-average molecu-

414

II 1 12

13

LONGANO

-I 1.4

FP

FIG. 3. Sedimentation equilibrium profile gastrin labeled gastrin at pH 2.5: ‘%I-labeled cpm, 3.5 fmol) was sedimented to equilibrium TL-100 ultracentrifuge at 15,000 rpm and 10°C h. The sample contained 5 mg/ml ovalbumin mM glycine, 10 mM Tris, 10 mM Na+ formate, mM Na+ acetate, pH 2.5.

lar weight of approximately gastrin aggregate. The Interaction

of ‘=I(11,000 in the for 48 and 10 and 10

89,000 for the

ET

AL.

The interaction between gastrin and transferrin at neutral pH was analyzed in more detail in a series of centrifugation experiments using different concentrations of gastrin and transferrin. The amount of free gastrin near the meniscus after centrifugation (55 min at 100,000 rpm and 37°C in the TL-100 ultracentrifuge) was determined as a function of the moles of gastrin bound per mole of transferrin (Fig. 5) and analyzed in the form of a Scatchard plot (inset). The best fit line obtained with the program LIGAND (25) assuming a single class of binding sites indicated a stoichiometry of 2.25 gastrin binding sites per transferrin molecule and an apparent binding constant of 6.4 PM. When the number of binding sites was constrained to 2, an apparent binding constant of 4.8 I.LM was obtained. To assess the possibility that the two sites had different affinities, the data were also fitted to a two-site model using the program LIGAND. The apparent binding constants obtained were 4.9 and 4.8 PM, suggesting that the two sites had identical affinity for gastrin. The interaction between gastrin and transferrin was significantly reduced by

of Gas&in and Tralzsfetin

Sedimentation equilibrium profiles for ‘251-labeled gastrin in the presence and absence of human transferrin are presented in Fig. 4. The solution conditions in Fig. 4a were 10 mM Hepes, pH 7.4, and 5 mg/ml ovalbumin. Under these conditions the addition of apotransferrin (12 PM) caused a marked increase in sedimentation of the labeled gastrin. The value of the weight-average molecular weight obtained from the slope of the In C/C,, vs r2 plot was 46,900. The addition of diferrictransferrin under the same conditions had little effect on the sedimentation properties of gastrin (Fig. 4a). The observation that cholecystokinins, which shares the same C-terminal pentapeptide as gastrin, but lacks gastrin’s pentaglutamate sequence, did not bind to apotransferrin (data not shown) suggested that gastrin was interacting with the anion binding site of transferrin.

-2-1.2

14

1.6

1.6

2

FIG. 4. The effect of iron saturation and salt concentration on the interaction between transferrin ‘l-labeled gastrin alone (A) and ‘l-labeled gastrin: (11,000 cpm, 3.5 fmol), or together with 12 @d apotransferrin (0), or 12 FM diferrictransferrin (U), was sedimented to equilibrium in the absence (a) and presence (b) of 150 mM NaCl. Conditions of sedimentation in the Airfuge were 33,000 rpm for 24 h at 10°C in 10 mM Na+ Hepes, pH 7.4, containing 5 mg/ml ovalbumin.

BINDING

OF GASTRIN

0 o 0

0.5 04

0

%1,0.3 0.2 JIM-’

JqrY!s2;

0

0.1 0 , O'* , Od

0

2

I6

8

IGI,

10

12

1L

,"i 16

0

0 I'"

18

, 20

2.0 22

j.iM

FIG. 5. Binding of gastrin and transferrin: The binding profile shows the moles of gastrin bound per mole of total transferrin (R) as a function of free gastrin (G) concentration. The inset represents a Scatchard plot of the data. Samples were centrifuged in 10 mM Na+ Hepes, pH 7.4, containing 5 mg/mi ovalbumin at 100,000 rpm and 3’7°C for 55 min, in the TL-100 ultracentrifuge.

TO TRANSFERRIN

415

valent crosslinking (12). A gastrin concentration of approximately 100 PM was required to inhibit the crosslinking reaction by 50%. However, because of the irreversibility of crosslinking the IC& value may be an overestimate of the & (26). We therefore decided to investigate the reversible interaction between gastrin and transferrin by sedimentation velocity and sedimentation equilibrium, to determine whether the Kd value thus obtained was low enough for the interaction to be physiologically relevant. The effects of added Fe3+ ions, and of pH, on the sedimentation behavior of gastrin were first investigated. Clear evidence was obtained for the formation of high-molecular-weight gastrin complexes in the presence of Fe3+ ions at pH 5.0 and at pH 7.4 (Table I, Fig. 1). Since complex formation was not observed in the absence of Fe3+ ions, these results provide direct evidence for an interaction between Fe3+ ions and gastrin. Interaction between gastrin and Cap+ and Mg2+ ions in trifluoroethanol (17), and between gastrin and Ca2’ ions in aqueous solution (16), has previously been observed by circular dichroic and NMR spectroscopy, respectively. Evi-

the addition of NaCl. The results in Table II refer to a series of sedimentation velocity experiments of mixtures of gastrin and transferrin prepared in solutions containing different concentrations of NaCl. In the absence of added NaCl the fraction of ‘251-labeled gastrin remaining near the meniscus was 11% of the original concentration. At higher concentrations of NaCl this fraction increased. At concentrations of 150 and 250 mM NaCl the fraction of the TABLE II labeled gastrin remaining at the meniscus THEEFFECTOFN~CICONCENTRATIONON was similar to that observed for a sample THE INTERACTION OF ‘%I-LABELED of gastrin alone. A complete sedimentaGASTRINAND TRANSFERRIN tion equilibrium profile for a mixture of lz51-labeled gastrin and apotransferrin in NaCl Apotransferrin ‘zI-iabeied gastrin 150 mM NaCl is presented in Fig. 4b. The at meniscus” (%) (PM) (FM) slope of the In C/C0 vs r2 plot for the 125111.0 + 0.6 0 12 labeled gastrin yielded values for the 29.5 2 0.6 25 12 weight-average molecular weight of 2600 50 12 47.9 +- 1.8 and 6300 in the absence and presence of 60.5 f 3.3 75 12 apotransferrin, respectively. These results 66.6 It 1.5 100 12 indicate that under these conditions ap72.6 k 2.5 150 12 proximately 5% of the gastrin was bound 12 72.9 + 1.0 250 to the transferrin. Experiments were not 0 0 72.4 k 2.0 performed at the pH of the gastric juice because of the aggregation and precipitaNote. Samples were sedimented in 10 mM Na+ Hepes, pH 7.4, 5 mg/ml ovalbumin at 100,000 rpm tion of gastrin itself at low pH (Fig. 2). DISCUSSION

An interaction between gastrin and transferrin was detected originally by co-

and 10°C for 55 min in the TL-100 ultracentrifuge. “The values represent the fraction of izI-labeled gastrim? in the top two lo-p1 fractions of the centrifuge tube at the conclusion of centrifugation. Values are the average of three samples + 1 SD.

416

LONGANO ET AL.

dence was also obtained for the formation of gastrin aggregates at pH values below 5 in the absence of added Fe3+ (Figs. 2 and 3). Since the molecular weight of ‘zI-[Nle15]-gastrin17 is 2205, the calculated weight-average molecular weight of 89,000 suggests that aggregates contained on average 40 molecules of gastrin. Sedimentation equilibrium experiments confirmed that gastrin and apotransferrin interact at pH 7.4 (Fig. 4). Moreover Scatchard analysis of the experimental data (Fig. 5) yielded a Kd of 6.4 PM, with approximately two binding sites per transferrin molecule. In contrast no interaction between gastrin and diferrictransferrin was detected. It is of course possible that other forms of gastrin might bind more tightly to apotransferrin than the ‘251-labeled [Nle15]-gastrin17 used in these experiments. Gastrin% is the predominant form of the hormone in serum and approximately half of the circulating gastrin% and gastrim, is sulfated on the sole tyrosine residue (23). Moreover NMR spectroscopy indicates that both cis and trans isomers of the proline of gastrim occur in solution (16). Finally iodination of the tyrosine residue of gastrim may reduce its affinity for transferrin, so that the experimental Kd of 6.4 PM is only an estimate of the strength of interaction in viva. What is the physiological relevance of the interaction between gastrin and apotransferrin in serum? Transferrin saturation in sera from seven healthy humans varied from 14 to 48% (mean 32.3%, SD 12.4%), with iron randomly distributed between the two binding sites (27). Since the normal serum transferrin concentration is 2.95 g/liter (37 PM) (28), the mean concentration of available binding sites is 37 X 2 X 0.677 = 50 PM. The ratio of bound to free gastrin will (for Kd = 6.4 PM) therefore be 50 f 6.4 = 7.8. In other words at a gastrin concentration similar to that of normal serum (25 pM (27)), and in the absence of other competing anions, nearly 90% of gastrin (22 PM) would be bound to transferrin. The data

presented in Table II and in Fig. 4b suggest that at physiological salt concentrations the interaction between gastrin and apotransferrin is considerably weaker. Nevertheless under the experimental conditions described in the legend to Fig. 4b (12 PM apotransferrin, 150 mM NaCl) approximately 5% of total gastrin was bound to apotransferrin. This residual gastrin-apotransferrin binding may perhaps explain the minor (0.9-2%) component of immunoreactive gastrin observed in the void volume when plasma samples from patients with the elevated serum gastrin levels characteristic of ZollingerEllison syndrome were chromatographed on Sephadex G-50 (29,30). A physiological role for the interaction between gastrin and transferrin in the uptake of dietary iron has been proposed previously (12). In this model gastrin and gastric transferrin are simultaneously released into the stomach lumen in response to the ingestion of food. Any ferric ions in the food are first complexed by gastrin and then transferred to transferrin, in the same way that anionic chelating agents like nitrilotriacetic acid catalyze transferrin loading in vitro (18). The loaded transferrin, which is presumably protected from proteolytic attack by the binding of ferric ions and/or gastrin, is then absorbed into the mucosal cells of the small intestine (11, 31). Some minor modifications are required to make the above model consistent with our present results. The aggregation and precipitation of gastrin at low pH (Fig. 2) suggest that chelation of ferric ions and interaction with transferrin may not occur until the pH of the gastric contents rises on entry into the small intestine. The presence of transferrin in bile (10) suggests that the liver and the gastric mucosa may both contribute to the transferrin observed in the small intestinal mucosa and further implies that transfer of ferric ions from gastrin to apotransferrin occurs predominantly in the lumen of the small intestine. It is also worth noting that the amounts of luminal gastrin (13 nmol/day in dogs (14)) and biliary transferrin (3.3 nmol/day in rats (10)) are of the same order. However, even after

BINDING

OF

GASTRIN

allowance for the difference in size between species, neither approaches the daily iron absorption of 20 pmol in humans, implying that transferrin must be recycled by the intestinal mucosa. The major results of this paper, that gastrin forms complexes with Fe3+ ions, and that the dissociation constant of the complex between gastrin and apotransferrin at low ionic strength is 6.4 PM, are both consistent with the hypothesis that gastrin and transferrin act synergistically in the uptake of dietary iron by the gastrointestinal tract. However, a direct involvement of the interaction between gastrin and transferrin in iron uptake cannot be concluded from the present study.

417

TO TRANSFERRIN MCKNIGHT,

G. S. (1986)

Proc Nat1 Acad Sci.

USA K&3723-3727. 11. HUEBERS, H. A., HUEBERS, E., CSIBA, E., RUMMEL, W., AND FINCH, C. A. (1983) Blood 61, 283-290. 12. BALDWIN, G. S., CHANDLER, R., AND WEINSTOCK, J. (1986) FEBS I.&t. 205.147-149. 13. SOLL, A. H., RODRIGO, R., AND FERRARI, J. C. (1981) Fed Proc 40.2519-2523. 14. JORDAN, P. H., AND YIP, B. S. S. C. (1972) Surgery 72,352-356. 15. UVNAS-WALLENSTEIN, K. (1977) Gastroenterology 73,487-491. 16. TORDA, A. E., BALDWIN, G. S., AND NORTON, R. S. (1985) Biochemistry 24,1720-1’727. 17. PEGGION, E., MAMMI, S., PALLJMBO, M., MORODER, L., AND WUNSCH, E. (1983) Biopolymers 22, 2443-2457. 18. BATES, G. W., AND SCHLABACH, M. R. (1973) J.

BioL Chem. 248,3228-3232. ACKNOWLEDGMENTS We thank Lin labeled gastrin.

Seet for

many

preparations

of ‘=I-

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