1CURE Digestive Diseases Research Center, Veterans Affairs Greater Los Angeles Health Care System, Los Angeles 90073 and Digestive Diseases Division, University of California Los Angeles School of Medicine, Los Angeles 90024; 2Pasarow Mass Spectrometry Laboratory, Departments of Psychiatry and Biobehavioral Sciences and Chemistry and Biochemistry, and the Neuropsychiatric Institute, University of California, Los Angeles, California 90095; and 3Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229
Submitted 15 January 2003 ; accepted in final form 17 September 2003
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ABSTRACT |
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cholecystokinin
Previous studies on the bioactivity of purified CCK-58 may be compromised by copurification of other bioactive peptides with CCK-58 or the copurification with inactive forms of CCK-58. Therefore, we synthesized rat CCK-58 (Fig. 1), demonstrated its purity, and used the purified peptide for evaluation of biological activity. Others (2) have proposed that the last seven amino acids contain all the information required for complete CCK activity and that the COOH-terminal tyrosine and phenylalanine amide are critical for biological activity. The availability of synthetic rat CCK-58 permits full chemical characterization of the peptide in advance of biological experiments.
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Prior studies (3, 11, 29) on the molecular forms of endocrine CCK involved formation of plasma as the first step in the processing of blood during the measurement of endocrine molecular forms and observed that CCK-33 was the major circulating form of CCK in humans. In addition, using similar methods that formed plasma as the first step in processing blood, others (9, 18) have detected only CCK-8 or CCK-22 or both in rat plasma. By contrast, our laboratory has reported that CCK-58 is a major circulating form in humans (6) and dogs (7) and that CCK-58 is the only endocrine form of CCK in rat (26).
The bioactivity of CCK-58 has been studied in several systems with purified peptide, and the results support the hypothesis that regions of CCK-58 outside the COOH terminus influence the expression of CCK activity (25). Purified canine CCK-58 was less potent than CCK-8 for stimulation of amylase release from isolated rat pancreatic acinar cells (25). Surprisingly, canine CCK-58 was more potent than CCK-8 for displacement of a labeled CCK-8 analog from mouse pancreatic membranes (28). CCK-8 and synthetic CCK-58 have different patterns of afferent nerve discharge (20). In addition, CCK-8 and purified porcine CCK-58 show different patterns of central catecholaminergic mechanisms and neuroendocrine functions (10).
In addition, synthetic canine CCK-58 stimulated rat bile-pancreatic volume, whereas CCK-8 caused no increase in volume (27), and purified porcine CCK-58 produced a pattern for contraction of the gallbladder different from that of CCK-8 (36). By contrast, porcine CCK-33 and CCK-8 had similar potencies for stimulation of amylase release from purified acinar cells (21). Furthermore, CCK-8 and CCK-33 had similar potency for displacement of an analog of CCK-10 label from purified rat acinar cell (4). Another group (34) has compared human CCK-33, porcine CCK-33, and CCK-8 for pancreatic secretion in vitro and in vivo. They showed that the peptides were equipotent in vitro, but the human CCK-33 peptide was about twofold more potent in vivo. This suggests that CCK-33 has the same binding and activation of the CCKA receptor as CCK-8, and the altered potency observed in the in vivo experiments probably resulted from differences in their circulating half-lives. The similar activity of CCK-8 and CCK-33 and the different activity of CCK-58 suggest that the 23-amino terminal residues of CCK-58 influence binding and activation of the CCKA receptor.
In this work, we describe the automated synthesis of rat CCK-58 and the purification and the biochemical analysis of the product. In addition, we compare CCK-8 and CCK-58 for immunoreactivity, binding to CCKA and CCKB receptors, and stimulation of pancreatic secretion in an in vivo anesthetized rat model. On the basis of these results and those of others, we propose that synthetic rat CCK-58 is an essential tool for future studies on the physiology of endocrine CCK.
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MATERIALS AND METHODS |
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Amino acid analysis synthetic sulfated rat CCK-58. A small aliquot of sulfated rat CCK-58 corresponding to 2 nmol based on absorbance was placed into a pyrolized hydrolysis tube. The peptide was hydrolyzed, and the resulting amino acids were quantitated as previously described (27).
Mass spectral analysis of rat CCK-58. A Perkin-Elmer Sciex (Thornhill, Canada) API III triple quadrupole mass spectrometer was tuned and calibrated as previously described (12). Vacuum centrifuge-dried HPLC fractions were redissolved in appropriate volumes of water-acetonitrile-formic acid (50/50/0.1, all by volume) and injected (10-20 µl/injection) into a stream of the same solvent entering the Ionspray ion source (10 µl/min). Spectra were obtained by scanning from m/z 300-2,000 (0.3-Da step size, 1-ms dwell time, 5.96 s/scan, orifice voltage 60) at an instrument resolution sufficient to resolve the isotopes of the polypropylene glycol per NH4+ singly charged calibrant ion at m/z 906 with 40% valley. Averaging of all the scans accrued from each sample injection and calculation of molecular weights from the series of multiple-charged ions found in the spectra was achieved with the MacSpec computer program (version 3.3, Perkin-Elmer Sciex). Deconvolution of the ion series into a molecular weight spectrum was achieved with the BioMultiView computer program (version 3.1.1, Perkin-Elmer Sciex).
Fast atom bombardment (FAB) spectra were obtained with a VG (Micromass) ZAB-SE instrument [8-kV accelerating potential, cesium bombardment at 22 kV and 2 uA, instrument mass resolution (M/M, 10% valley) of 1,000] using metanitrobenzyl alcohol as matrix. Matrix-assisted laser desorption ionization (MALDI) mass spectra were obtained on a time-of-flight mass spectrometer (ABI Voyager DE-STR) using
-cyano-4-hydroxycinnamic acid as matrix.
CCK RIA. Standard curves were made with CCK-8, CCK-22, CCK-33, and CCK-58, which were quantitated by amino acid analysis or absorbance at 280 nm. Antiserum RO16 (raised to sulfated CCK-10) reacts equally well with CCK-8, CCK-22, and CCK-33 (IC50 = 5, 7, and 5 pM, respectively), but this antiserum is about sixfold less potent for canine CCK-58 (IC50 = 32 pM). Antiserum RO16 does not require a sulfate group on the tyrosine seven amino acids from the COOH terminus for high-affinity binding of CCK analogs (37). This antisera has negligible cross-reactivity with gastrin peptides and reacts well with oxidized CCKs (37). Antibody RO16 was used at a 1:100,000 dilution. The label used was porcine CCK-33 radiolabeled with 125I-Bolton-Hunter reagent. Fractions from the HPLC were dried by vacuum centrifugation to 10-50 µl. Fractions and standard curve peptides (CCK-8 and CCK-58) were diluted to 600 µl with RIA buffer [0.1 M sodium phosphate buffer, pH 7.5, containing 0.05 M NaCl and 0.025 M disodium EDTA, 0.1% wt/vol RIA grade bovine serum albumin and 0.1% Triton X-100 (Sigma, St. Louis MO)]. The 600-µl sample was vortexed for 10 s and allowed to set at room temperature for 30 min. Then 200 µl antisera RO16 in RIA buffer and 200 µl buffer containing 3,000 counts/min (cpm) of labeled CCK-33 were added simultaneously to samples in 600 µl. The final 1-ml volume was incubated with the radiolabel for 16 h at 4°C, and then the free and bound radiolabels were separated with charcoal previously equilibrated with Dextran-70 for 16 h.
Binding of CCK-8 and CCK-58 to CCKA and CCKB receptors expressed in Chinese hamster ovary (CHO) cells. Binding experiments were performed on intact CHO cells transfected with rat CCKA [a gift from Dr. L. Miller (8)] and rat CCKB receptors [The cDNA was a kind gift from Dr. S. Wank, and it was transfected into CHO cells (19)]. 125I-lableled BH-CCK-8 (2,000 Ci/mmol) purchased from Amersham Biosciences (Piscataway, NJ) was used as the radioligand for both cell lines. Cells were cultured in poly-L-lysine-coated 24-well plates to a final density of 2 x 106 cells/well in DME/F12 medium containing 10% fetal bovine serum. Cell monolayer was rinsed twice with 1 x PBS followed by adding 1 ml binding buffer (Waymouth's medium, 20 mM HEPES, pH 7.4, 0.1% bacitracin, and 0.2% bovine serum albumin) to each well. Binding reactions were started by adding 125I-labeled BH-CCK-8 (20-40 pM) in the presence of increasing concentrations of nonlabeled CCK peptides as indicated. After 1-h incubation at 4°C, cells were rinsed twice with ice-cold 1 x PBS and dissolved in 1 ml 1% Triton X-100. Radioactivities in cell lysate (bound) and medium (free) were counted, and the results were expressed as percent maximal binding (with tracer alone).
Statistical analysis. Binding competition and dose-response curves were analyzed using Prism 3.0 program (GraphPad, San Diego, CA). Significance of difference was determined using the Student's unpaired t-test. When more than two groups were compared, significance was determined by one-way ANOVA (Statistix, Miami, FL).
Bioassay of synthetic rat CCK-58. Exocrine pancreatic secretory responses to synthetic rat CCK-58 and CCK-8 were determined by a modification of previously described methods (35). Male Sprague-Dawley rats (280-400 g body wt, fasted for 20-24 h), were anesthetized with Inactin (thiobutabarbital sodium salt, Research Biochemicals International, MA) 100 mg/kg ip. The right jugular vein (PE-50, Intramedic) was catheterized; intravenous fluids were delivered at an infusion rate 1.08 ml/h. The trachea was intubated to facilitate breathing and to prevent aspiration (PE-260, Intramedic). Through a midline celiotomy, the common bile-pancreatic duct was catheterized (PE-10, Intramedic) for collection of mixed bile-pancreatic juice. Collections were begun after a 2-h postsurgical period of stabilization. Collections were made into 1-ml syringes at 15-min intervals. The duodenum was canulated (PE-50, Intramedic) at the level of the sphincter of Oddi. Previously collected bile-pancreatic juice diluted 1:1 with saline was infused into the duodenum at 1.08 ml/h to prevent release of endogenous CCK. CCK-8 and CCK-58 were infused at 10, 30, and 90 pmol·kg-1·h.
After collecting unstimulated bile-pancreatic secretion for 1 h, either CCK-8 or synthetic CCK-58 was administered intravenously for 2 h. Only one dose was given to each rat. Peptides were prepared by diluting stock solutions into 0.15 M NaCl containing 0.1% (wt/vol) BSA (RIA grade, Sigma). Other animals received only vehicle and served as controls for any time-dependent changes in unstimulated secretion. Volume of collected bile-pancreatic juice was measured using a Hamilton syringe to the nearest 0.001 ml. Amylase was determined on each 15-min sample by the method of Bernfeld (1).
Data analysis. Statistical analyses were performed on the incremental differences between the average output per 15 min of basal measurements and the average output per 15 min during the final 90 min measurements (plateau level) after either vehicle, CCK-8, or CCK-58 infusion. Significance of differences was determined using the Student's unpaired t-test (Sigma Stat SPSS Science, Chicago, IL). Results are presented as means ± SE.
Molecular forms of CCK in rat blood. Casein was gavaged into three Sprague-Dawley rats (300 g) that had been fasted overnight. For each rat, after 30 min, the animal was anesthetized with 10 mg/kg pentabarbitol sodium. Then the heart was exposed by blunt dissection, and 9 ml of blood were withdrawn by cardiac puncture into EDTA tubes containing 3,000-10,000 cpm each of I125-labeled CCK-58 and 10 ml of cold 0.3 ammonium acetate (pH 3.6) containing 100 µg each of enzyme inhibitors from Peptide International (Louisville, KY): diprotin A, E64D, aprotinin, Ac-SIMP-1, and antipain and 10 µg of (1-48) CCK-58. Less than 20 s after end of cardiac puncture, 80 ml ice-cold 0.3 M ammonium acetate (pH 3.6) containing 0.5 M NaCl and enzyme inhibitors at the same concentration as described above were added to the 19 ml of the blood-buffer sample, and this 99-ml mixture was centrifuged at 3,000 g for 10 min to remove red blood cells. The 0.5 M NaCl was used to help prevent red blood cell lysis in the acidic buffer.
The supernatant was loaded onto a C-18 SepPak (10 g Millipore) equilibrated in 0.1% trifluoroacetate, rinsed with 20 ml 0.1% trifluoroacetate, and then eluted with 50 ml 50% acetonitrile containing 0.1% trifluoroacetate. Four-milliliter fractions collected during the loading, rinse, and elution steps were evaluated for I125-labeled CCK-58 recovery by gamma counting. For two rats, all eluted fractions were pooled until 10 min after the I125-labeled CCK-58 radioactive fractions. In one rat, I125-labeled CCK-8 was added to the mixture before adding rat blood. In this rat, the radioactivity between the I125-labeled CCK-8 and I125-labeled CCK-58 was pooled for the HPLC step. Both methods gave essentially the same result. The pooled fractions eluted from SepPak were diluted sixfold and loaded onto an analytical Vydac C-18 reverse-phase HPLC column (10 µm, 4.6 x 250 mm, part number 218TP104; Western Analytical, Hesperia CA) equilibrated in 0.1% trifluoroacetate. The sample was eluted with a 10-min gradient to 20% acetonitrile and then a 120-min gradient to 35% acetonitrile. Two-minute fractions were dried by vacuum centrifuge, and then counted for evaluation of radioactivity recovery. Label and antibody were added simultaneously in immunoassay buffer and then vortexed to dissolve the samples. The results for three rats are presented as means ± SE.
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RESULTS |
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The dried synthetic peptide with blocking groups-resin mixture weighed 930 mg. The crude peptide produced by cleaving and unblocking the peptide in trifluoroacetate was 340 mg. Figure 2 shows the final purification step of sulfated rat CCK-58. HPCE of the purified peptide showed that it was 87% pure (Fig. 3). The amino acid analysis of rat CCK-58 is shown in Table 1.
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Mass spectral analysis of rat CCK-58. Under the described electrospray ionization (ESI) conditions samples of sulfated CCK that were 92% pure by HPCE criteria typically gave signals for sulfated and desulfated CCK in about a 60/40 ratio. The proportion of desulfated CCK was significantly increased at elevated orifice voltages. Extensive desulfation of sulfated CCK was also observed when the samples were analyzed by FAB and MALDI ionization. Desulaftion during ESI was less than that observed during FAB and surprisingly less than what was observed during MALDI.
Immunoreactivity of sulfated rat CCK-58. Figure 4 shows that rat CCK-58 was one-fifth as potent as CCK-8 for displacement of Bolton-Hunter CCK-33 label from antiserum RO16 (this antiserum binds to the COOH-terminal region CCK). These data suggest that the COOH terminus of synthetic rat CCK-58 has a tertiary structure that differs from the conformation of the same region of CCK-8. Rat CCK-8, CCK-22, and CCK-33 do not appear to have the same tertiary structure as CCK-58, because their IC50's are 5, 7, and 5 (compared with 32 pM for CCK-58).
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Bioactivity of sulfated rat CCK-58. The volume of combined bile-pancreatic juice was not significantly increased by any dose of CCK-8, whereas CCK-58 significantly increased bile-pancreatic juice volume at doses of 10 pmol·kg-1·h (from basal of 314 ± 21 to 362 ± 17) and 30 pmol·kg-1·h (from basal of 325 ± 20 to 392 ± 13). For amylase secretion, rat CCK-58 is significantly more efficacious than CCK-8 at 10 and 30 pmol·kg-1·h and tends to be more efficacious at 90 pmol·kg-1·h (Fig. 6). These results could reflect the fact that CCK-58 is at higher concentrations because of its longer circulating half-life (15). However, the significantly increased bile-pancreatic fluid secretion stimulated by CCK-58 at 90 pmol·kg-1·h (Fig. 7) is not a function of different bioavailability. For example, higher concentrations of CCK-8 actually caused less fluid secretion than observed for the dose of 90 pmol·kg-1·h with CCK-58. The average (>2-h period of CCK-8 or CCK-58 infusion) incremental output of amylase and fluid are shown in Fig. 8.
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Molecular forms of CCK in rat blood. In an attempt to identify the endocrine molecular forms of CCK, our laboratory systematically evaluated the recovery and stability of exogenous radiolabeled CCK-58 during all steps of blood processing (26). We observed that radiolabeled CCK-58 was extensively degraded if plasma was formed as the first step in processing of rat blood (26). Because this step of blood processing was the first step used by others in previous studies that detected only CCK-8 and CCK-22 in rat plasma (9, 18), it is possible that larger forms of CCK were degraded during plasma formation during previous attempts to characterize the endocrine forms of rat CCK.
This new optimized method, shown to prevent loss of CCK-58 (26), detected only CCK-58 in rat blood (Fig. 9) when CCK release was stimulated with casein. Radiolabeled CCK-58 eluted at 108 min (Fig. 9). The immunoreactive peak eluting just after labeled CCK-58 is endogenous CCK-58. The concentration of CCK-58 in casein-stimulated rat blood was 15 ± 5 pM. This pattern of elution was observed when radiolabeled CCK-58 and unlabeled CCK-58 were added to fasted rat blood (26).
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DISCUSSION |
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In a previous in vivo model of CCK bioactivity, natural canine CCK-58 was equipotent to CCK-8 for decreasing gastric motor function, but the longer peptide had a significantly longer duration of action than the octapeptide (24). In another in vivo model where recordings from jejunal mesenteric afferent bundles were recorded, the peak discharge pattern was different for CCK-8 and CCK-58, the peak discharge frequency was higher for CCK-8, but the duration of response was longer for CCK-58. Preliminary data in an awake rat model (39) and results reported here in an anesthetized rat model show the same pattern: the anticipated increase in amylase secretion with CCK-8 and the unexpected result that only CCK-58 stimulates fluid. It has not been determined whether the stimulation of bile-pancreatic secretion in the present model is regulated by CCKA or CCKB receptors.
In these studies, CCK-8 displaced label more potently from CCKB receptors than from CCKA receptors. In contrast, others have shown (16) that CCK-8 displaces label more potently from CCKA receptors than from CCKB receptors on guinea pig pancreatic acinar cells. Furthermore, Saito et al. (30) have shown that radiolabeled CCK-33 is displaced from CCKA and CCKB membrane receptors by CCK-8 with the same potency. These differences in order of potency for CCK-8 between CCKA and CCKB receptors could be caused by the label used, the cellular context of the receptor (native cells or transfected CHO cells), or the use of membranes instead of whole cell preparations (31, 32).
The molecular forms of CCK in rat blood were determined by a new method that prevented degradation of radiolabeled CCK-58 (26). This method yielded >85% recoveries for all suggested molecular forms of rat CCK (CCK-8, CCK-22, CCK-33, and CCK-58) and used a CCK-specific RIA with standards for each form of rat CCK (26). When plasma is formed immediately after rat blood was drawn, the plasma frozen, thawed, purified by stepwise solid-phase chromatography, and chromatographed by reverse-phase HPLC, 50% of radiolabeled CCK-58 added to blood was degraded (data not shown). By contrast, if blood is acidified with buffer containing protease inhibitors and radiolabeled CCK-58 is added to blood and the blood is processed using the same method employed in this work, a single peak of radioactivity is observed eluting in the same position as 125I-labeled CCK-58. These data suggest that proteolytic enzymes cleave CCK-58 during processing of blood when plasma is formed in the absence of proteolytic inhibitors. Furthermore, CCK-58 is the only form observed if exogenous canine CCK-58 is infused into dogs and blood is drawn after infusion of CCK-58 had stopped (V. Eysselein, personal communication), suggesting that CCK-58 is not degraded in vivo. Therefore, we hypothesize that CCK-58 is degraded by proteolytic enzymes activated during collection and processing of blood.
CCK-58 was the major endocrine form of CCK detected in dog (7) and human (6), but smaller molecular forms of CCK were also observed. However, in these previous studies (6, 7) no radiolabeled CCK-58 was available for evaluation of endogenous peptide stability. If degradation of CCK-58 was fully inhibited in the human and dog studies, then CCK-58 might have been the only detectable endocrine form of CCK in these species.
Production of CCK-33, using FMOC-Tyr-sulfate and FMOC chemistry (23), served as a prototype for the synthesis of rat CCK-58. The peptide was synthesized with FMOC chemistry instead of t-BOC chemistry so that the incorporated sulfated tyrosine would experience gentler unblocking conditions (89% trifluoroacetate instead of hydrogen fluoride), which reduced the desulfation of the sulfated tyrosine. Attempts to maximize sulfation while minimizing the presence of blocking groups resulted in our using 1-h unblocking times at room temperature. The purification strategy used for sulfated CCK-58 was analysis of each HPLC fraction by HPCE (a technique orthogonal to reverse-phase chromatography HPLC). This strategy enabled the pooling of the most homogeneous fractions.
Antisera RO16 was produced against CCK-10, and the antiserum has negligible cross-reactivity with gastrin (37). Sulfated rat CCK-58 was sixfold less immunoreactive than CCK-8 with antiserum RO16 (IC50 = 32 and 5 pM, respectively). This antiserum does not require a sulfate group and is directed toward the COOH terminus of CCK. Furthermore, this antibody is fully active with CCK-8, suggesting that the antibody binds with amino acids around positions 50-53 in CCK-58. The fact that dog (25), pig (14), and now rat CCK-58 peptides have decreased immunoreactivity with a variety of antiserum suggests that the COOH-terminal epitope is influenced by the amino terminus of the peptide. This concept is supported by the fact that both bioactivity (14, 25) and immunoreactivity (14, 38) of CCK-58 can be increased by trypsin digestion of CCK-58. The decreased immunoreactivity of COOH terminally directed antibodies suggests that the abundance of CCK-58 may have been underestimated in studies on the molecular forms of CCK in tissue and plasma (5, 29). The varied bioactivity and immunoreactivity of CCK-58 relative to CCK-8 demonstrates the importance of having a CCK-58 standard for measurements of the endogenous forms of CCK.
The studies reported here were designed to evaluate the physiological relevance of endocrine CCK-58. CCK-58 is the only detectable molecular form present in rat blood if its release is stimulated by a trypsin inhibitor (26) or by protein (present studies). Therefore, evaluating this molecular form is essential for understanding the endocrine actions of CCK.
In vitro binding experiments are useful for determining the pharmacology of an agonist at its receptor. For the CCKA receptor, three systems are available for binding studies: 1) acinar cells from rat, 2) gallbladder muscle strips or gallbladder cells from a species other than rat, or 3) cells transfected with the rat CCKA receptor. Acinar cell preparations were eliminated because they degrade 125I-labeled CCK-58 under radioreceptor assay conditions. Gallbladder preparations were not considered because the rat does not have a gallbladder, and it is known that the species of receptor is very important for expression CCKA biological activity (17). Miller's laboratory demonstrated that rat CCKA receptors transfected into CHO cells are indistinguishable from CCKA receptors on rat pancreatic acinar cells, especially for binding of agonists and antagonists (13). Therefore, until the degradation of agonist in rat acinar cell preparations can be inhibited, the transfected CHO cell is a reasonable alternate physiological model. These binding experiments demonstrated that CCK-8 was more potent than CCK-58 for displacing CCK-8 radiolabel.
Finally, CCK-58 caused a different pattern of biological activity from CCK-8 (stimulation of fluid secretion) in an in vivo model. We used the anesthetized model in this work because it is used more widely than the awake rat model. This choice is supported because preliminary data show the same pattern of pancreatic secretion in the awake rat (39) as in the anesthetized model.
With the use of a new reagent (synthetic CCK-58), we have observed two important facts: CCK-58 has qualitative differences in bioactivity compared with other molecular forms of CCK in vitro and in vivo, and CCK-58 is the only molecular form of CCK detected in rat blood when CCK was stimulated by casein. The abundance and differing activity of CCK-58 compared with smaller forms suggest that this molecular form is an important reagent for determining the physiology, receptor interactions, and intracellular second messenger activation of CCK.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-33850 and the CURE Digestive Diseases Center Grant DK-41301 and utilized the Peptide Biochemistry and Molecular Probes and the Animal Cores and the Medical Research Service of the Veterans Health Service. The National Science Foundation and National Institutes of Health Shared Instrument programs and the W. M. Keck Foundation provided funding for purchase of the mass spectrometers (to K. Faull).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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