NH2-terminal of gastrin-17 in duodenal ulcer disease: identification of progastrin-17

Serum gastrin concentrations were measured using antisera with specificity for the carboxyl and amino terminus of gastrin-17 in 50 healthy subjects and 18 patients with active duodenal ulcer disease (DU). The amino terminal of gastrin-17 immunoreactivity was significantly higher in DU patients than in healthy subjects. NH2-terminus of gastrin-17 immunopurified material from serum of DU patients was subjected to Sephadex G50 column chromatography and eluates were monitored by an additional antiserum EG10 that recognizes COOH-terminally extended gastrin. Besides the NH2 terminal tridecapeptide of gastrin-17, COOH-terminally extended progastrin was found. This may reflect abnormal processing of gastrin in patients with active duodenal ulcer disease.     

Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein

Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.     

Amino terminal fragments of human progastrin from gastrinoma

Two peptides which copurified from a human gastrinoma were found to correspond to the amino acid sequence deduced for the amino terminal portion of human and porcine progastrin. The sequence of peptide A is Ser-Trp-Lys-Pro-Arg-Ser-Gln-Gln-Pro-Asp-Ala-Pro-Leu-Gly-Thr-Gly-Ala-Asn- Arg-Asp-Leu-Glu-Leu which is identical to an amino terminal portion of human progastrin. The sequence of peptide. B is identical to that of peptide A except it is missing the first five amino acids. If peptide A corresponds to the amino terminus of progastrin, the signal peptidase cleaves at an Ala-Ser bond.     

Is helodermin-like immunoreactivity in human thyroid C cells due to a salmon calcitonin-like substance?

Helodermin-like and salmon calcitonin (sCT)-like immunoreactivities co-existed in a subset of human calcitonin (hCT)-containing cells in normal human thyroid tissue and medullary thyroid carcinomas. Helodermin/sCT-immunoreactive cells were mostly different from calcitonin gene-related peptide (CGRP)-positive cells. Helodermin and sCT immunoreactivities were not identified in pulmonary and pancreatic hCT-positive neuroendocrine tumors, except for a few lung tumor cells showing positive staining with one of two sCT antisera used. Helodermin immunoreactivity demonstrated by rabbit antiserum R0086 was completely abolished in the presence of synthetic sCT, while sCT immunoreactivity was not absorbed by synthetic helodermin. The carboxyl terminal Arg30-Thr31 sequence (and Pro35 amide structure) of helodermin would be the epitopic site recognized by this antiserum, since a similar amino acid sequence is present in sCT molecules but absent from hCT and CGRP.     

The constitution and properties of phosphorylated and unphosphorylated C-terminal fragments of progastrin from dog and ferret antrum

Antibodies to the extreme C-terminal tryptic (nona-) peptide fragment of porcine progastrin have been used in radioimmunoassay to identify progastrin fragments in dog, ferret and pig antral mucosa extracts and to monitor their purification. In addition to previously characterised phosphorylated and unphosphorylated C-terminal tryptic peptides of porcine progastrin a minor form corresponding to the C-terminal octapeptide (i.e. des-Ser C-terminal nonapeptide) was isolated and characterised. The latter form together with phosphorylated and unphosphorylated forms of the nonapeptides were also isolated and chemically characterised from dog antrum, and the unphosphorylated nonapeptide was characterised from ferret antrum. The primary amino acid sequences of the dog, ferret and pig nonapeptides were identical. In ferret the unphosphorylated nonapeptide predominated, and in dog the phosphorylated form predominated; in pig both forms of the nonapeptide were well represented. Intact progastrin was identified in gel filtration eluates of extracts of all 3 species, but occurred only in relatively low concentrations. The nonapeptides did not stimulate acid secretion in the conscious gastric fistula rat and they did not modify the acid response to G17. Phosphorylation of progastrin-derived peptides is evidently well conserved across a range of species even though there appear to be differences in the relative proportions of phosphorylated and unphosphorylated forms.     

Neurotensin-like immunoreactivity generated by pepsin from human plasma and gastric tissue

The present study demonstrates precursors of neurotensin-like immunoreactivity (NTLI) endogenous to human gastric tissue and plasma, and the existence of a gastric NTLI-generating enzyme system. The molecular size of the NTLI-precursors in plasma and gastric tissue were estimated by gel permeation chromatography to be ca 50,000-60,000 and 60,000-70,000 Da, respectively. The neurotensin-like peptide generated from the precursor was detected with a carboxyl-terminally directed antiserum but did not cross-react with an amino-terminally directed antiserum. A neurotensin-like peptide isolated from pepsin-treated human plasma was characterized by mass spectrometry and its amino acid sequence determined. This novel nonapeptide, referred to as kinetensin, failed to affect pentagastrin-stimulated acid secretion or blood pressure in the rat. Sequence homologies between neurotensin, kinetensin and proteins of the serum albumin family suggest a common evolutionary origin and raise questions regarding albumin-like proteins as precursors of regulatory peptides.     

Peptides in rat brain immunoreactive for the carboxyl terminal extension of cholecystokinin: distribution and chromatography

Utilizing an antiserum raised against a peptide fragment identical to part of the carboxyl terminal extension of cholecystokinin (CCK) predicted by the sequence of CCK mRNA [7], an antiserum has been generated which does not detect CCK 39, CCK 33, CCK 8, CCK 4 or gastrin 171. This antiserum detects several peptides in rat brain, one similar in size to CCK 33 and another slightly larger than CCK 8. These peptides may represent carboxyl-terminally extended forms of CCK, though their chemical structure has not been determined. These peptides are present in all brain regions where CCK 8 can be detected. The abundance of these peptides, their localization in CCK terminal regions, and their enrichment in synaptosome preparations [1] imply that the tryptic cleavage and amidation reaction occur late in the processing of CCK (as has been observed for other biologically active peptides), and probably occur in the synaptic vesicle.     

Isolation and microsequence analysis of a novel form of neuromedin U from guinea pig small intestine

A multidimensional chromatographic regimen has been used to isolate and purify a peptide showing immunoreactivity for neuromedin U from guinea pig small intestine. Microsequence Edman N-terminal analysis and C-terminal analysis by enzymatic digestion showed this peptide to be a nonapeptide with the following sequence: H-Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2. The C-terminal octapeptide of this sequence is the same as porcine NMU-8, and the C-terminal heptapeptide is identical to rat NMU(17-23).     

Progastrin-like immunoreactivity in porcine antrum: identification and characterization with region-specific antisera

In an effort to identify and characterize precursors of gastrin in tissues, we generated region-specific antisera against a synthetic progastrin peptide, Try-Gly-Trp-Met-Asp-Phe-Gly-Arg-Arg (GL9), as deduced from the nucleotide sequence of gastrin mRNA. This antisera did not cross-react with gastrin or progastrin peptides with shorter carboxyl-terminal extensions. Progastrin-like immunoreactivity (PGLI) was measured in porcine antrum at a concentration of 6.8 +/- 1.2 pmol/g wet weight (mean +/- SE, n = 5), or roughly 0.2% of that of gastrin. On Sephadex G50 chromatography, a major peak of PGLI was eluted as a slightly larger molecule than gastrin heptadecapeptide (G17) but possessed the same N-terminal immunoreactivity. These findings suggest that G17 may be formed by processing of a carboxyl-terminally extended precursor as an alternative to cleavage of big gastrin (G34).     

Immunohistochemical localization to pyloric antral G cells of peptides derived from porcine preprogastrin

Antibodies to the peptides (designated cryptic A and B) that flank the G34 region of pig progastrin were used in immunohistochemical studies of the gastrointestinal tract. In elution and restaining experiments, the same cells were revealed by the cryptic peptide antibodies, and by antibodies specific for C-terminus of G17 and N-terminus of G34. The cells reacting with the cryptic peptide antibodies were localized predominantly to antral mucosa. They were found in pig, ferret, dog and cat but not in man, guinea pig, rat or mouse; presumably in the latter species there are amino acid substitutions in the cryptic peptides that influence immunoreactivity with the present antibodies. The results indicate that progastrin production occurs only in G cells in the gut, and that a single population of cells produces all the predicted regions of progastrin.     

Partial structure of a large canine cholecystokinin (CCK58): Amino acid sequence

A cholecystokinin molecule larger than any previously chemically characterized was purified from canine proximal small intestine mucosa. The purification procedure consisted of sequential steps of affinity chromatography, gel filtration, and high pressure liquid chromatography. Activity was detected and quantitated by radioimmunoassay with an antibody that recognized the carboxyl terminal sequence of porcine cholecystokinin. Microsequencing of the purified peptide revealed an amino terminal nonadecapeptide sequence (AQKVNSGEPRAHLGALLAR) not present in known cholecystokinin molecules followed by a nonadecapeptide sequence (YIQQARKAPSGRMSVIKNL) that corresponds exactly to the amino terminal sequence of porcine cholecystokinin 39 except for reversed positions of a Met and a Val residue. Based on the sequence analysis, immunoreactivity, and presence of biological activity in two bioassay systems, this peptide, tentatively named cholecystokinin 58, may be a biosynthetic precursor of the smaller forms previously characterized in gastrointestinal and brain tissues.     

Identification and characterization of glycine-extended post-translational processing intermediates of progastrin in porcine stomach

We developed a radioimmunoassay specific for glycine-extended progastrin processing intermediates (G-Gly) using antisera generated against the synthetic peptide Tyr-Gly-Trp-Met-Asp-Phe-Gly. Distribution of immunoreactivity in the porcine gastrointestinal tract obtained with this antibody paralleled that of gastrin with the mucosa containing the highest quantity, 116 +/- 22 pmol/g, wet weight (mean +/- S.E., n = 5), or roughly 4% of gastrin concentration. This immunoreactivity was localized specifically to antral mucosal G-cells by immunohistochemistry. On Sephadex G-50 column chromatography of porcine antral mucosal extracts glycine-extended progastrin processing intermediates were separated into three principal molecular forms, each corresponding to known molecular forms of gastrin, component I, tetratriacontagastrin (G34) and heptadecagastrin (G17). Following purification by antibody-coupled affinity chromatography, one molecular form corresponding to G17 in size was shown to have an amino terminus identical to that of G17. Another molecular form corresponding to G34 in size could be converted to the molecular form corresponding to G17 by tryptic digestion. Our findings indicate that glycine-extended progastrin processing intermediates may serve as immediate precursors for each molecular form of gastrin, thus suggesting an alternative pathway for gastrin biosynthesis more complex than that previously conceived.     

Protein kinase C phosphorylates the synthetic peptide Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val in the presence of phospholipid plus either Ca2+ or a phorbol ester tumor promoter

The synthetic nonapeptide Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val is a substrate for in vitro phosphorylation by a partially purified preparation of rat brain protein kinase C, with Kmapp of about 130 microM. The closely related peptide kemptide was a much weaker substrate, bovine serum albumin was not a substrate and the peptide Arg-Arg-Lys-Ala-Ala-Gly-Pro-Pro-Val was a weak inhibitor of the enzyme. Protein kinase C-catalyzed phosphorylation of histone III-S and the nonapeptide are regulated by identical mechanisms since with both substrates the reaction required added phospholipid and either Ca2+ (1mM) or TPA (200 nM TPA). Our findings show that polypeptides containing multiple basic residues followed by the sequence Ala-Ser can be substrates for TPA-stimulated phosphorylation by protein kinase C.     

Identification by specific radioimmunoassay of two novel peptides derived from the C-terminus of porcine preprogastrin

A radioimmunoassay has been developed using antibodies to a synthetic analogue of the C-terminal hexapeptide sequence of the porcine gastrin precursor. Boiling water extracts of porcine antral mucosa contained immunoreactive material that diluted in parallel with standard peptide. Concentrations of immunoreactivity were 5.5 +/- 0.8 nmol X g-1 (mean +/- S.E.M.) in antral mucosa and were closely similar to those of C-terminal heptadecapeptide gastrin immunoreactivity (5.0 +/- 0.6 nmol X g-1). Approximately 30-fold lower concentrations were found in porcine duodenum. A similar distribution was found in ferret, but human, rat and chicken antrum did not contain significant quantities of immunoreactivity. Gel filtration of porcine antral extracts on Sephadex G-50 revealed a single peak of immunoreactivity eluting in a similar position to G17, but on anion-exchange chromatography two peaks of immunoreactive material were separated. These also differed in their retention time on reverse phase HPLC. Both peptides are probably derived by tryptic cleavage at the C-terminus of porcine preprogastrin. No evidence was found to suggest that there are significant quantities of unprocessed preprogastrin in hog antral mucosa. The precise chemical difference between the two immunoreactive peptides identified here remains to be established; together, however, they provide specific markers for progastrin synthesis.     

Isolation and characterization of the porcine hypothalamic growth hormone releasing factor

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 2500 porcine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and 2 steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analyses of the intact peptide and its carboxyl terminal cyanogen bromide digestion fragment. Reverse phase liquid chromatography of the native peptide and synthetic replicates showed that the molecule possesses an amide rather than a free acid at its carboxyl terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Arg-Val-Arg-Leu-NH2 using approximately 6 nmol of material.     

Characterization of the C-terminal flanking peptide of human beta-preprotachykinin

The nucleotide sequence of cDNA encoding the common biosynthetic precursor of substance P, neurokinin A and neuropeptide K (beta-preprotachykinin) predicts that, in the human, the precursor contains a C-terminal flanking peptide of 19 amino acid residues [beta-preprotachykinin(111-129)-peptide]. Using an antiserum raised against synthetic human beta-preprotachykinin(117-126)-peptide in radioimmunoassay, we have demonstrated that an extract of a human neuroendocrine tumor of the adrenal medulla contained approximately equimolar concentrations of C-terminal preprotachykinin immunoreactivity (C-PPT-IR), substance P and neurokinin A. The C-terminal preprotachykinin flanking peptide was purified to homogeneity and its primary structure was determined. The amino acid sequence of the peptide, Ala-Leu-Asn-Ser-Val-Ala-Tyr-Glu-Arg-Ser-Ala-Met-Gln-Asn-Tyr-Glu, indicates identity with beta-preprotachykinin(111-126)-peptide. The data suggest that the C-terminal flanking peptide, like the tachykinins, is packed into secretory storage vesicles but the Arg127-Arg128-Arg129 residues in human beta-preprotachykinin are removed from the peptide by the action of endogenous processing enzyme(s).     

Isolation and sequence analysis of human bombesin-like peptides

The decapeptide form of human gastrin releasing peptide was isolated from acid extracts of liver tissue containing a metastatic human bronchial carcinoid tumor. A larger form also was isolated and partially characterized. During gel permeation chromatography the major immunoreactive peak eluted in the same region as synthetic gastrin releasing decapeptide while a second minor immunoreactive peak eluted near gastrin releasing peptide. Bombesin-like immunoreactivity (BLI) was purified by successive applications to reverse phase high pressure liquid chromatography (HPLC) columns. After four successive HPLC purifications a single peak of bombesin-like immunoreactivity was detected. Amino acid analysis, microsequence analysis and coelution with synthetic peptide indicated that the predominant form present in metastatic tumor tissue was identical to the decapeptide form of canine gastrin-releasing peptide. The less abundant form was purified by cation exchange chromatography followed by reverse phase high pressure liquid chromatography. Partial microsequence analysis of this peptide, through the first 11 residues, was Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu. This sequence differed from that of hog heptacosapeptide gastrin releasing peptide at positions 1,3,4 and 5 and from the canine peptide as positions 1,3,5, and 7.     

Isolation and characterization of a neurotensin-like decapeptide from a canine upper small intestinal extract

Neurotensin-like immunoreactivity can be detected in extracts of canine upper gastrointestinal mucosa when measured by carboxyl terminal but not by amino terminal antibodies to neurotensin. The nature of this immunoreactive material was characterized by complete purification on gel filtration and HPLC followed by peptide microsequence analysis. The structure obtained was Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-(Leu), identical in structure to the carboxyl terminal decapeptide of neurotensin. It cannot, however, be excluded that this neurotensin decapeptide was generated from a larger neurotensin-like peptide during the extraction procedure by a physiological or artificial enzymatic process. Since carboxyl terminal neurotensin fragments containing eight or more residues have full biological activity, this peptide may be responsible for neurotensin-like biological activities within the mucosa of, or after release from, the upper gut.     

Crucial role of position 40 for interactions of CCK-58 revealed by sequence of cat CCK-58

Evidence suggests that amino terminal extensions of CCK-8 affect the carboxyl terminal bioactive region of CCK. Cat CCK-58 was purified by low pressure reverse phase and ion-exchange chromatography steps and several reverse phase HPLC steps. The purified peptide and its tryptic fragments were characterized by mass spectral analysis and microsequence analysis. The structure of cat CCK-58 is: AVQKVDGEPRAHLGALLARYIQQARKAPSGRMSVIKNLQSLDPSHRISDRDY(SO3) MGWMDF-amide. Cat and dog CCK-58 are identical except for position 40 which is serine in cat and asparagine in dog. Radioimmunoassay detected cat CCK-58 about 1/10th as well as dog CCK-58, indicating a marked effect on C-terminal immunoreactivity. Cat CCK-58 with a serine at position 40, the same residue found in pig, mouse, cow and rabbit CCK-58, can be used as a unique bioprobe for defining how amino terminal amino acids influence the structure and bioactivity of the carboxyl terminal region of CCK.     

Identification of xenin, a xenopsin-related peptide, in the human gastric mucosa and its effect on exocrine pancreatic secretion.

One of the peptides previously discovered in amphibians is the octapeptide xenopsin. As immunohistochemistry has also indicated the presence of xenopsin immunoreactivity in man, we extracted in the present investigation xenopsin-immunoreactive material from human gastric mucosa and purified it to homogeneity with several high performance liquid chromatography (HPLC) reverse phase and ion exchange chromatographic steps. The eluates were monitored with a radioimmunoassay for amphibian xenopsin. Determination of the amino acid sequence revealed a 25-amino acid peptide having 6 C-terminal amino acids in common with amphibian xenopsin. The sequence of this peptide, termed xenin 25, is M-L-T-K-F-E-T-K-S-A-R-V-K-G-L-S-F-H-P-K-R-P-W-I-L. The peptide was custom-synthesized. Mass spectrometry of the synthetic and the extracted peptide revealed identical molecular mass. Purification of 250 ml of human postprandial plasma with Sep-Pak C18 cartridges, reverse phase HPLC, and ion exchange chromatography demonstrated circulating xenin immunoreactivity at a retention time identical to xenin 25. The amount of xenin immunoreactivity at the position of xenin 25 on C18-HPLC increased significantly after a meal. A radioimmunoassay utilizing antibodies to xenin 25 and a 125I-labeled analogue of xenin 25 was used to measure immunoreactive xenin in the plasma of 10 volunteers. There was a significant rise of xenin immunoreactivity in the plasma after a meal. Intravenous infusion of the synthetic peptide in dogs stimulated exocrine pancreatic secretion beginning at a dose of 4 pmol/kg/min. The maximal effect was seen with 64 pmol/kg/min. We have detected, therefore, a new peptide, xenin 25, in human gastric mucosa; we have provided evidence for the presence of this peptide in the human circulation, and have shown a rise of plasma xenin concentrations after a meal. This peptide stimulates exocrine pancreatic secretion. Its physiologic role deserves further investigation.