Vol. 131. No. 6 PrmtPd in 1I.S.A.
Distribution Secretory
of Cholecystokinin Granule Subtypes*
C. M. TURKELSON
AND
Forms
J. HAMILTON
Veterans Administration Medical Center, Kansas City, Missouri (C. M. T.) and Biochemistry (J. H.), University of Kansas Medical
64128; and the Departments of Physiology Center, Kansas City, Kansas 66103
ABSTRACT
buoyant densities, suggesting that they contain a greater number of osmotically active molecules than granules of lower density and that short or all intragranular CCK forms are osmotically active. CCK secretory granules also contained novel CCK forms; in addition to previously characterized forms, granules contained a CCK form that appears to be CCK-6 and a form that could arise from cleavage of CCK-58 at position 4, 10, or 12. Because intragranular enzymes are responsible for peptide posttranslational processing, the intragranular CCK forms observed in the present study are likely to be authentic CCK-processing products. Finally, CCK sorting in intestine apparently differs from that in a rat medullary thyroid carcinoma cell line, in which CCK-22 and CCK-33 are not found in the regulated secretory pathway. (Endocrinology 131: 2533-2539,1992)
Peptides and proteins destined to be released in response to stimuli are found in the regulated secretory pathway. Substances in this pathway are packaged into secretory granules, wherein they are often rendered osmotically inactive by complexing to an oppositely charged molecule. The complexing mechanism employed by members of the cholecystokinin (CCK) peptide family is unknown, but the heterogenous charges of CCK peptides makes it possible that different CCK peptides have different abilities to form intragranular complexes. If the number of osmotically active intragranular CCK peptides varies, corresponding variations in secretory granule density should result, and when intestinal CCK secretory granules were purified on isotonic density gradients, four granule peaks were observed. Granules containing greater proportions of short CCK forms tended to have the lowest
P
destined to be secreted in response to stimuli are packaged into secretory granules at the Golgi, wherein they undergo posttranslational processing (see Ref. 1 for review) and often condense into insoluble secretory granule cores (2). This condensation keeps the granule con-
zymes appear to be altered in response to these changes. For example, insulin posttranslational processing appears to involve two endopeptidases that cleave between adjacent basic amino acids; the activity of one of these enzymes is enhanced by low levels of calcium, and its pH optimum is near neutrality, suggesting that it is likely to be more active at or near the Golgi. Similar considerations suggest that the other enzyme is more likely to be active in the more acidic secretory granules, wherein the calcium concentration is sufficient to activate an enzyme that requires high calcium concentrations for maximal activity (7). Like insulin, POMC is cleaved between adjacent basic amino acids by an enzyme that may be calcium activated and may have an acidic pH optimum (8). Further, the enzyme involved in POMC processing and at least one of the enzymes involved in insulin processing appear to be related to the KEX2 gene product of yeast (9, 10, 11). This latter enzyme, which cleaves at adjacent basic amino acids in the yeast pro-a-factor to form the peptidemating pheromone, is also calcium activated (12; see Ref. 13 for review). CCK processing may not exclusively involve a KEX2-like gene product. In intestinal mucosa, only CCK-33 is formed by cleavage at adjacent basic amino acids, and the remaining chemically characterized major CCK forms (CCK-58, CCK39, CCK-22, and CCK-8) appear to result from cleavages of larger peptides at single basic amino acid residues, sites not extensively cleaved by the KEX2 gene product (13). However, it is important to note that identification of CCK-processing products has almost always been conducted by analyzing CCK extracted from unfractionated tissue, and it is, therefore, possible that not all of the chemically characterized CCK forms are located within secretory granules. Indeed, one
EPTIDES
tents
in
an
isoosmotic
in Intestinal
relationship
with
the
surrounding
cytoplasm (3) and prevents these highly concentrated substances from leaking out of the granules. Often, peptides condense by complexing with other substances. Insulin, which exists in granules in concentrations sufficient to cause it to crystalize, complexes with Zn2+ (4,5), and other peptides may complex to the acidic chromogranins or secretogranins (6). In both examples, complexing is based upon the attraction of substances with opposite electrostatic charges. However, certain peptides may present secretory granules with unusual storage problems. Among these are the members of the cholecystokinin (CCK) peptide family, which are found in intestine in long basic forms and short acidic forms; should these heterogeneously charged forms have different abilities to form intragranular complexes, the number of osmotically active particles in CCK granules containing different proportions of differently charged forms should differ and produce alterations in secretory granule density. One purpose of the present investigation was to assess this possibility. As peptides leave the Golgi, they also become subjected to an increasingly acidic environment and increased calcium concentrations, and the activities of peptide-processing enReceived April 24, 1992. Address correspondence and requests for reprints to: C. M. Turkelson, Veterans Administration Medical Center, Research (151), 4801 Linwood Boulevard, Kansas City, Missouri 64128. * This work was supported by the Research Service of the V.A.
2533
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 01:24 For personal use only. No other uses without permission. . All rights reserved.
2534
CCK
SECRETORY
report suggests that CCK-33 and CCK-22 are not packaged into secretory granules in a rat medullary thyroid carcinoma cell line (14). Thus, a second purpose of the present investigation was to gather information on intragranular CCKprocessing enzymes by determining which CCK forms are found within rat intestinal secretory granules.
Materials Secretory granule
and Methods
purification
The upper one fourth of the intestine was removed from decapitated 200. to 250-g male rats allowed ad libitum access to food and water and flushed with 50 ml cold 0.15 M saline and 50 ml saline containing 1 mM dithiothreitol to remove contents and mucous. These procedures were approved by the local institutional committee on the care and use of animals. The intestinal piece was then slit lengthwise and placed in cold 0.25 M sucrose, 50 mM Tris, 1 rnM MgCl2, and 0.25% (wt/vol) BSA, pH 8.0 (STMB buffer), and the mucosa was scraped off the muscularis with a glass microscope slide. Subsequent procedures were performed at 4 C unless otherwise noted. The solution and mucosal scrapings were centrifuged at 200 X g for 3 min, and the supernatant was discarded. The pellet was brought to 20 ml with STMB buffer, homogenized with a Dounce homogenizer (10 strokes with the loose and 10 strokes with the tight pestle; Wheton, Millville, NJ), and centrifuged at 800 x g for 10 min. Pilot studies demonstrated that it was necessary to include BSA in the homogenization buffer to reliably obtain 800 x g pellets. The resulting supernatant was set aside, and the pellet was resuspended with 15 ml STMB buffer, and homogenized and centrifuged as described above. The supernatants from the two 800 x g centrifugations were then combined and centrifuged at 3,000 X g for 15 min to remove additional debris. This supernatant was then centrifuged at 10,000 X g for 15 min to yield a crude secretory granule pellet that was brought to 2.5 ml with STMB buffer and resuspended with five strokes of the Dounce homogenizer loose pestle. One-milliliter portions were then layered over discontinuous Nycodenz (Accurate Chemical and Scientific Corp., Westbury, NY) gradients consisting of 1.5 ml 9.2% (wt/vol), 1.5 ml 13.8%, 1.5 ml 18%, 2.0 ml 20%, 2.0 ml 22%, and 1.5 ml 27.6% Nycodenz. Nycodenz solutions were buffered at pH 8.0 with 10 rnM Tris, and were made isotonic with sucrose, as described by the supplier. Gradients were then centrifuged at 38,000 rpm for 2 h in a Beckman SW40Ti rotor (Palo Alto, CA). Gradients were fractionated by upward displacement at a flow rate of 0.375 ml/min. One-minute fractions were collected. Electron microscopy demonstrated the presence of secretory granules in all fractions that contained immunoreactive CCK that entered the Nycodenz gradients. Density gradient fractions were prepared for RIA by diluting a portion of each fraction with an equal volume of 4 N acetic acid containing 2% (vol/vol) Triton X-100. Diluted fractions were then placed in a boiling water bath for 10 min to inactivate intragranular enzymes that metabolized the RIA tracer and resulted in aberrantly high levels of apparent CCK immunoreactivity (15), chilled, neutralized with sodium hydroxide, diluted with RIA buffer (16), and assayed. Although it is established that acid efficiently extracts large CCK forms from tissue (17, 18), sulfated CCK-8 is poorly recovered with certain acid extractions (19). Therefore, the synthetic sulfated octapeptide was added to various granule preparations, and its recovery assessed; recoveries were 70.7 + 4.7% (mean f SE; n = 5) from crude secretory granule pellets and 89.6 + 4.0% (n = 10) from granules purified on density gradients,
RIA RIA was conducted as previously described (20), R015 at a titer of 1:32,500. The tracer for the RIA was CCK-33 (Peninsula Laboratories, Belmont, CA) labeled Hunter reagent (New England Nuclear, Boston, MA). 5 ~1 0.05 N acetic acid) and 17 ~1 0.05 M borate buffer, added to a vial containing [‘251]Bolton-Hunter reagent
using antiserum synthetic porcine with [‘*‘I]BoltonCCK-33 (5 pg in pH 10.0, were (the benzene in
Endo. 1992 Vol 131 .No 6
GRANULES
which the labeled Bolton-Hunter reagent was supplied had been previously evaporated under a gentle stream of N2). The mixture was incubated for 1 h in an ice bath oscillating at 120 cycles/min, and then for 3 h at 4 C. Thereafter, 0.5 ml 6 M guanidine HCl was added to the vial, which was incubated for 5 min at 4 C. The entire contents of the vial were purified by reverse phase HPLC. Purification was accomplished by eluting an analytical Vydac C Is column (The Separations Group, Hesperia, CA) for 20 min at 1 ml/min with 30% (vol/voI) acetonitrile containing 0.1% (wt/vol) trifluoroacetic acid (TFA) and then for 50 min with 35% acetonitrile containing 0.1% TFA. The radioactive peak eluting at 55 min was used as tracer. The sensitivity of the RIA using this tracer was 0.25 PM, and it had an ED,, of 5 PM. The cross-reactivities of antiserum ROl5 with various synthetic CCK forms, determined as previously described (16), are shown in Table 1.
Other assays Protein was assayed using the Bio-Rad (Richmond, CA) assay. An aliquot of the homogenate was prepared for protein assay by adding it to BSA-free homogenization buffer and centrifuging it at 40,000 x g for 15 min. Protein in the resulting pellet was solubilized by overnight incubation in 1 N sodium hydroxide at 4 C and diluted with water before assay. Gradient fractions were similarly treated with sodium hydroxide and diluted before assay. The mitochondrial marker cytochrome c oxidase was assayed as described by Cooperstein and Lazarow (21), and the lysosomal marker enzyme P-glucuronidase was assayed as described by Levy and Conchie (22).
Chromatography Reverse phase HPLC of granule preparations was performed using a column similar to the one described above. After injecting samples onto the column, the column was washed at a flow rate of 1 ml/min for 5 min with 5% (vol/vol) acetonitrile containing 0.1% (wt/vol) TFA and then brought to 20% acetonitrile containing 0.1% TFA over a 5-min period. Samples were then eluted at 1 ml/min with a linear gradient of 20-40% acetonitrile in 0.1% TFA over 80 min. Natural porcine CCK-39 for column calibration was obtained from V. Mutt, Karolinska Institute (Stockholm, Sweden), and natural canine CCK-58 was a gift from J. R. Reeve, Jr., Center for Ulcer Research and Education (Los Angeles, CA). Other details regarding HPLC were as described previously (19). Density gradient fractions and resuspended crude (10,000 X g) secretory granule pellets were prepared for HPLC by adding them to an equal volume of 4 N acetic acid containing 2% Triton X-100. The resulting mixtures were then incubated overnight at 4 C, and debris and detergent were removed from the samples using a CIR cartridge (19). Acetonitrile was evaporated off the cartridge eluates under N1 at 37 C, and the product was applied to the HPLC column. Preliminary studies similar to those previously described (19) were conducted to insure that CCK was not metabolized into smaller fragments by intragranular or contaminating enzymes during these procedures. In these studies synthetic CCK-33 was incubated overnight at 4 C with secretory granules diluted with acetic acid containing detergent, purified on Cla cartridges, and then chromatographed by HPLC, as described above. These results demonstrated that CCK was not metabolized or degraded during extraction by showing that incubated CCK-33 eluted as a single peak with the mobility of intact CCK-33.
TABLE synthetic
1. Percent CCK forms
Peptide CCK-4 CCK-5 CCK-6 CCK-7 CCK-8(s)
cross-reactivities and gastrin
% Cross-reaction 1 2 170 107 100
CCK-8(s), The sulfated fated amidated octapeptide; peptide; hG-171, nonsulfated
of antiserum
Peptide
R015 with % Cross-reaction
CCK-8(ns) CCK-8(0H) CCK-33 hG-I71 amidated octapeptide; CCK-8(0H), sulfated human gastrin-17.
157
Distribution Secretory
of Cholecystokinin Granule Subtypes*
C. M. TURKELSON
AND
Forms
J. HAMILTON
Veterans Administration Medical Center, Kansas City, Missouri (C. M. T.) and Biochemistry (J. H.), University of Kansas Medical
64128; and the Departments of Physiology Center, Kansas City, Kansas 66103
ABSTRACT
buoyant densities, suggesting that they contain a greater number of osmotically active molecules than granules of lower density and that short or all intragranular CCK forms are osmotically active. CCK secretory granules also contained novel CCK forms; in addition to previously characterized forms, granules contained a CCK form that appears to be CCK-6 and a form that could arise from cleavage of CCK-58 at position 4, 10, or 12. Because intragranular enzymes are responsible for peptide posttranslational processing, the intragranular CCK forms observed in the present study are likely to be authentic CCK-processing products. Finally, CCK sorting in intestine apparently differs from that in a rat medullary thyroid carcinoma cell line, in which CCK-22 and CCK-33 are not found in the regulated secretory pathway. (Endocrinology 131: 2533-2539,1992)
Peptides and proteins destined to be released in response to stimuli are found in the regulated secretory pathway. Substances in this pathway are packaged into secretory granules, wherein they are often rendered osmotically inactive by complexing to an oppositely charged molecule. The complexing mechanism employed by members of the cholecystokinin (CCK) peptide family is unknown, but the heterogenous charges of CCK peptides makes it possible that different CCK peptides have different abilities to form intragranular complexes. If the number of osmotically active intragranular CCK peptides varies, corresponding variations in secretory granule density should result, and when intestinal CCK secretory granules were purified on isotonic density gradients, four granule peaks were observed. Granules containing greater proportions of short CCK forms tended to have the lowest
P
destined to be secreted in response to stimuli are packaged into secretory granules at the Golgi, wherein they undergo posttranslational processing (see Ref. 1 for review) and often condense into insoluble secretory granule cores (2). This condensation keeps the granule con-
zymes appear to be altered in response to these changes. For example, insulin posttranslational processing appears to involve two endopeptidases that cleave between adjacent basic amino acids; the activity of one of these enzymes is enhanced by low levels of calcium, and its pH optimum is near neutrality, suggesting that it is likely to be more active at or near the Golgi. Similar considerations suggest that the other enzyme is more likely to be active in the more acidic secretory granules, wherein the calcium concentration is sufficient to activate an enzyme that requires high calcium concentrations for maximal activity (7). Like insulin, POMC is cleaved between adjacent basic amino acids by an enzyme that may be calcium activated and may have an acidic pH optimum (8). Further, the enzyme involved in POMC processing and at least one of the enzymes involved in insulin processing appear to be related to the KEX2 gene product of yeast (9, 10, 11). This latter enzyme, which cleaves at adjacent basic amino acids in the yeast pro-a-factor to form the peptidemating pheromone, is also calcium activated (12; see Ref. 13 for review). CCK processing may not exclusively involve a KEX2-like gene product. In intestinal mucosa, only CCK-33 is formed by cleavage at adjacent basic amino acids, and the remaining chemically characterized major CCK forms (CCK-58, CCK39, CCK-22, and CCK-8) appear to result from cleavages of larger peptides at single basic amino acid residues, sites not extensively cleaved by the KEX2 gene product (13). However, it is important to note that identification of CCK-processing products has almost always been conducted by analyzing CCK extracted from unfractionated tissue, and it is, therefore, possible that not all of the chemically characterized CCK forms are located within secretory granules. Indeed, one
EPTIDES
tents
in
an
isoosmotic
in Intestinal
relationship
with
the
surrounding
cytoplasm (3) and prevents these highly concentrated substances from leaking out of the granules. Often, peptides condense by complexing with other substances. Insulin, which exists in granules in concentrations sufficient to cause it to crystalize, complexes with Zn2+ (4,5), and other peptides may complex to the acidic chromogranins or secretogranins (6). In both examples, complexing is based upon the attraction of substances with opposite electrostatic charges. However, certain peptides may present secretory granules with unusual storage problems. Among these are the members of the cholecystokinin (CCK) peptide family, which are found in intestine in long basic forms and short acidic forms; should these heterogeneously charged forms have different abilities to form intragranular complexes, the number of osmotically active particles in CCK granules containing different proportions of differently charged forms should differ and produce alterations in secretory granule density. One purpose of the present investigation was to assess this possibility. As peptides leave the Golgi, they also become subjected to an increasingly acidic environment and increased calcium concentrations, and the activities of peptide-processing enReceived April 24, 1992. Address correspondence and requests for reprints to: C. M. Turkelson, Veterans Administration Medical Center, Research (151), 4801 Linwood Boulevard, Kansas City, Missouri 64128. * This work was supported by the Research Service of the V.A.
2533
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 01:24 For personal use only. No other uses without permission. . All rights reserved.
2534
CCK
SECRETORY
report suggests that CCK-33 and CCK-22 are not packaged into secretory granules in a rat medullary thyroid carcinoma cell line (14). Thus, a second purpose of the present investigation was to gather information on intragranular CCKprocessing enzymes by determining which CCK forms are found within rat intestinal secretory granules.
Materials Secretory granule
and Methods
purification
The upper one fourth of the intestine was removed from decapitated 200. to 250-g male rats allowed ad libitum access to food and water and flushed with 50 ml cold 0.15 M saline and 50 ml saline containing 1 mM dithiothreitol to remove contents and mucous. These procedures were approved by the local institutional committee on the care and use of animals. The intestinal piece was then slit lengthwise and placed in cold 0.25 M sucrose, 50 mM Tris, 1 rnM MgCl2, and 0.25% (wt/vol) BSA, pH 8.0 (STMB buffer), and the mucosa was scraped off the muscularis with a glass microscope slide. Subsequent procedures were performed at 4 C unless otherwise noted. The solution and mucosal scrapings were centrifuged at 200 X g for 3 min, and the supernatant was discarded. The pellet was brought to 20 ml with STMB buffer, homogenized with a Dounce homogenizer (10 strokes with the loose and 10 strokes with the tight pestle; Wheton, Millville, NJ), and centrifuged at 800 x g for 10 min. Pilot studies demonstrated that it was necessary to include BSA in the homogenization buffer to reliably obtain 800 x g pellets. The resulting supernatant was set aside, and the pellet was resuspended with 15 ml STMB buffer, and homogenized and centrifuged as described above. The supernatants from the two 800 x g centrifugations were then combined and centrifuged at 3,000 X g for 15 min to remove additional debris. This supernatant was then centrifuged at 10,000 X g for 15 min to yield a crude secretory granule pellet that was brought to 2.5 ml with STMB buffer and resuspended with five strokes of the Dounce homogenizer loose pestle. One-milliliter portions were then layered over discontinuous Nycodenz (Accurate Chemical and Scientific Corp., Westbury, NY) gradients consisting of 1.5 ml 9.2% (wt/vol), 1.5 ml 13.8%, 1.5 ml 18%, 2.0 ml 20%, 2.0 ml 22%, and 1.5 ml 27.6% Nycodenz. Nycodenz solutions were buffered at pH 8.0 with 10 rnM Tris, and were made isotonic with sucrose, as described by the supplier. Gradients were then centrifuged at 38,000 rpm for 2 h in a Beckman SW40Ti rotor (Palo Alto, CA). Gradients were fractionated by upward displacement at a flow rate of 0.375 ml/min. One-minute fractions were collected. Electron microscopy demonstrated the presence of secretory granules in all fractions that contained immunoreactive CCK that entered the Nycodenz gradients. Density gradient fractions were prepared for RIA by diluting a portion of each fraction with an equal volume of 4 N acetic acid containing 2% (vol/vol) Triton X-100. Diluted fractions were then placed in a boiling water bath for 10 min to inactivate intragranular enzymes that metabolized the RIA tracer and resulted in aberrantly high levels of apparent CCK immunoreactivity (15), chilled, neutralized with sodium hydroxide, diluted with RIA buffer (16), and assayed. Although it is established that acid efficiently extracts large CCK forms from tissue (17, 18), sulfated CCK-8 is poorly recovered with certain acid extractions (19). Therefore, the synthetic sulfated octapeptide was added to various granule preparations, and its recovery assessed; recoveries were 70.7 + 4.7% (mean f SE; n = 5) from crude secretory granule pellets and 89.6 + 4.0% (n = 10) from granules purified on density gradients,
RIA RIA was conducted as previously described (20), R015 at a titer of 1:32,500. The tracer for the RIA was CCK-33 (Peninsula Laboratories, Belmont, CA) labeled Hunter reagent (New England Nuclear, Boston, MA). 5 ~1 0.05 N acetic acid) and 17 ~1 0.05 M borate buffer, added to a vial containing [‘251]Bolton-Hunter reagent
using antiserum synthetic porcine with [‘*‘I]BoltonCCK-33 (5 pg in pH 10.0, were (the benzene in
Endo. 1992 Vol 131 .No 6
GRANULES
which the labeled Bolton-Hunter reagent was supplied had been previously evaporated under a gentle stream of N2). The mixture was incubated for 1 h in an ice bath oscillating at 120 cycles/min, and then for 3 h at 4 C. Thereafter, 0.5 ml 6 M guanidine HCl was added to the vial, which was incubated for 5 min at 4 C. The entire contents of the vial were purified by reverse phase HPLC. Purification was accomplished by eluting an analytical Vydac C Is column (The Separations Group, Hesperia, CA) for 20 min at 1 ml/min with 30% (vol/voI) acetonitrile containing 0.1% (wt/vol) trifluoroacetic acid (TFA) and then for 50 min with 35% acetonitrile containing 0.1% TFA. The radioactive peak eluting at 55 min was used as tracer. The sensitivity of the RIA using this tracer was 0.25 PM, and it had an ED,, of 5 PM. The cross-reactivities of antiserum ROl5 with various synthetic CCK forms, determined as previously described (16), are shown in Table 1.
Other assays Protein was assayed using the Bio-Rad (Richmond, CA) assay. An aliquot of the homogenate was prepared for protein assay by adding it to BSA-free homogenization buffer and centrifuging it at 40,000 x g for 15 min. Protein in the resulting pellet was solubilized by overnight incubation in 1 N sodium hydroxide at 4 C and diluted with water before assay. Gradient fractions were similarly treated with sodium hydroxide and diluted before assay. The mitochondrial marker cytochrome c oxidase was assayed as described by Cooperstein and Lazarow (21), and the lysosomal marker enzyme P-glucuronidase was assayed as described by Levy and Conchie (22).
Chromatography Reverse phase HPLC of granule preparations was performed using a column similar to the one described above. After injecting samples onto the column, the column was washed at a flow rate of 1 ml/min for 5 min with 5% (vol/vol) acetonitrile containing 0.1% (wt/vol) TFA and then brought to 20% acetonitrile containing 0.1% TFA over a 5-min period. Samples were then eluted at 1 ml/min with a linear gradient of 20-40% acetonitrile in 0.1% TFA over 80 min. Natural porcine CCK-39 for column calibration was obtained from V. Mutt, Karolinska Institute (Stockholm, Sweden), and natural canine CCK-58 was a gift from J. R. Reeve, Jr., Center for Ulcer Research and Education (Los Angeles, CA). Other details regarding HPLC were as described previously (19). Density gradient fractions and resuspended crude (10,000 X g) secretory granule pellets were prepared for HPLC by adding them to an equal volume of 4 N acetic acid containing 2% Triton X-100. The resulting mixtures were then incubated overnight at 4 C, and debris and detergent were removed from the samples using a CIR cartridge (19). Acetonitrile was evaporated off the cartridge eluates under N1 at 37 C, and the product was applied to the HPLC column. Preliminary studies similar to those previously described (19) were conducted to insure that CCK was not metabolized into smaller fragments by intragranular or contaminating enzymes during these procedures. In these studies synthetic CCK-33 was incubated overnight at 4 C with secretory granules diluted with acetic acid containing detergent, purified on Cla cartridges, and then chromatographed by HPLC, as described above. These results demonstrated that CCK was not metabolized or degraded during extraction by showing that incubated CCK-33 eluted as a single peak with the mobility of intact CCK-33.
TABLE synthetic
1. Percent CCK forms
Peptide CCK-4 CCK-5 CCK-6 CCK-7 CCK-8(s)
cross-reactivities and gastrin
% Cross-reaction 1 2 170 107 100
CCK-8(s), The sulfated fated amidated octapeptide; peptide; hG-171, nonsulfated
of antiserum
Peptide
R015 with % Cross-reaction
CCK-8(ns) CCK-8(0H) CCK-33 hG-I71 amidated octapeptide; CCK-8(0H), sulfated human gastrin-17.
157
