Cloning and developmental expression analysis of prokineticin 2 and its receptor PKR2 in the Syrian hamster surpachiasmatic nucleus.

Prokineticin 2 (PK2) and its receptor (PKR2) may play important roles in transmitting circadian rhythm information from the suprachiasmatic nucleus (SCN) to downstream neural targets. In this study, we identified PK2 and PKR2 genes in the Syrian hamster through a homologous cloning approach by performing 5' and 3'-RACE. Sequence alignments indicate significant homology between the mouse, rat and Syrian hamster sequences. In situ hybridization experiments indicate that PK2 and PKR2 are expressed in the SCN, with the expression of PK2 with a pronounced amplitude change across the daily cycle under DD conditions. Recent studies indicate that the molecular machinery of the central clock in the suprachiasmatic nucleus only mature gradually during development. We thus studied the developmental expression of PK2 and PKR2 in the surpachiasmatic nuclei of the perinatal animals. PKR2 is expressed in the Syrian hamster SCN at all time points examined, while the expression of PK2 remains undetectable at prenatal time points. Expression of PK2 begins to be detectable on postnatal day 3 and onwards on day 5 and day 7. The developmental appearance of the PK2 expression rhythm may reflect the maturation process of the central clock's molecular machinery.

Isolation and characterization of four VIP-related peptides from red-bellied newt, Cynops pyrrhogaster.

Four novel bioactive peptides were isolated from the red-bellied newt, Cynops pyrrhogaster, using a bioassay system monitoring the rectum contraction of the Japanese quail, Coturnix japonica. As these peptides are structurally related to vasoactive intestinal polypeptide (VIP), we termed these peptides newt VIP-related peptides 1, 2, 3, and 4 (NVRP-1, -2, -3, and -4). The primary sequences of these peptides were determined to be HSDAVFTDNYSRLLGKTALKNYLDGALKKE (NVRP-1), HSDAVFTDNYSRLLAKTALKNYLDGALKKE (NVRP-2), HSDAVFT-DNYSRLLGKIALKNYLDEALKKE (NVRP-3), and HSDAVFTDNYSRLLGKT-ALKNYLDSALKKE (NVRP-4). The N-terminal regions of these NVRPs possessed homology at the amino-acid level to various VIP, while the NVRP C-termini differed from VIPs significantly. All of the VIP consist of 28 amino-acid residues with amidated forms at the C-termini, whereas NVRPs possess 30 amino-acid residues and have free forms at the C-termini. NVRPs exert relaxant activities on isolated quail rectums in a dose-dependent manner, with threshold concentrations between 1 x 10(-8) and 3 x 10(-8) M. NVRPs also exhibited potent relaxant activities acting on the newt duodenum at 3 x 10(-8) M. As yet, this is the first isolation of biologically active VIP-related peptides from urodele.

Native and recombinant proguanylin feature identical biophysical properties and are monomeric in solution.

Guanylin, an intestinal peptide hormone and endogenous ligand of guanylyl cyclase C, is produced as the corresponding prohormone proguanylin. The mature hormone consists of 15 amino acid residues, representing the COOH-terminal part of the prohormone comprised of 94 amino acid residues. Here we report the recombinant expression and purification of proguanylin with its native disulfide connectivity, as well as the biophysical characterization of the recombinant and native protein. The comparison of recombinant and native proguanylin revealed identical biophysical and structural properties, as deduced from CZE, HPLC, and mass spectrometry, as well as NMR spectroscopy and CD spectroscopy at various temperatures and pH values. Exhaustive analytical ultracentrifugation studies were employed for protein concentrations up to the millimolar range to determine the association state of recombinant as well as native proguanylin, revealing both proteins to be monomeric at the applied solution conditions. As a result, a former identified close proximity between the termini of proguanylin is due to intramolecular interactions.

Characterization of dopuin, a polypeptide with special residue distributions.

A 62-residue polypeptide, dopuin, has been isolated from pig small intestine. It is distinguished by an N-terminal part with a high content of proline (7 in a 26-residue segment), a C-terminal part with a high proportion of histidine (3 in a 9-residue segment), and six half-cystine residues in three intrachain disulphide bridges (connecting positions 22-25, 23-54 and 35-44). The Cys and Pro distributions suggest a tight and special conformation. In contrast to PEC-60 and somatostatin, it has no established inhibitory effect on insulin secretion. At 10 nM concentration, a weak inhibitory tendency is less than half of that of the other two peptides. Like gastrointestinal trefoil peptides, dopuin has three disulphide bridges, Ala-Pro segments, and many charged residues, but they are differently distributed and dopuin belongs to a separate, apparently novel family. However, dopuin is similar to a peptide corresponding to an expressed-sequence-tag cDNA of human fetal liver and spleen, establishing the nature of the mature form of the product of this cDNA, and showing a general tissue, age, and species distribution of this peptide. A truncated form of vimentin, composed of its C-terminal 37 residues, vimentin-C37, was also purified and structurally characterized. These two peptides increase the complexity of known intestinal polypeptides and at least dopuin has properties compatible with specific biofunctions.

Characterization of the endogenous intestinal peptide that stimulates the rectal gland of Scyliorhinus canicula.

It has been postulated that gut peptides play a major role in the regulation of rectal gland secretion in elasmobranchs. An isolated perfused rectal gland secretion in elasmobranchs. An isolated perfused rectal gland preparation was developed for Scyliorhinus canicula that responded to dibutyryl 3',5'-cyclic monophosphate plus 3-isobutyl-1-methylxanthine, increasing chloride clearance rates threefold over basal levels. Activity was stimulated by an endogenous peptide, isolated in pur form by reverse-phase high-performance liquid chromatography from the intestine of S. canicula. The primary structure was established as Ser-Pro-Ser-Asn-Ser-Lys-Cys-Pro-Asp-Gly-Pro-Asp-Cys-Phe-Val-Gly-Leu-Met- NH2. This is a sequence identical to that of the tachykinin scyliorhinin II. Perfusion of synthetic scyliorhinin II increased secretion rate in the rectal gland of S. canicula in a dose-dependent manner with a maximal response at 10(-6) M, whereas vasoactive intestinal peptide, a stimulator in the spiny dogfish, Squalus acanthias, had no effect. We propose that scyliorhinin II is the uncharacterized peptide rectin, previously identified from the intestine of S. canicula.

Characterization of the pancreatic hormones from the Brockmann body of the tilapia: implications for islet xenograft studies.

The Brockmann body of the teleost fish, the tilapia (Oreochromis nilotica) has been considered as a potential source of islet xenograft tissue for patients with insulin-dependent diabetes. This study describes the purification from an extract of tilapia Brockmann bodies of insulin and several peptides arising from different pathways of post-translational processing of two proglucagons, two prosomatostatins and proPYY. The primary structure of tilapia insulin is similar to insulins from other teleosts (particularly the anglerfish, Lophius americanus) except that the strongly conserved glutamine residue at position 5 in the A-chain, a residue that is important in the binding of insulin to its receptor, is replaced by glutamic acid. In common with other teleosts, the tilapia Brockmann body expresses two non-allelic glucagon genes. Alternative pathways of post-translational processing lead to glucagons with 29 and 36 amino acid residues derived from proglucagon I and glucagons with 29 and 32 residues derived from proglucagon II. Glucagon-like peptides with 30 and 34 residues derived from proglucagon II were also isolated. In each case, the longer peptide is a C-terminally extended form of the shorter. Tilapia peptide tyrosine-tyrosine (PYY) was isolated in a C-terminally alpha-amidated from with 36 amino acid residues that is structurally similar (89% sequence identity) to anglerfish PYY. A 30-amino acid peptide, representing the C-terminal flanking peptide of PYY, was also isolated that shows only 53% sequence identity with the corresponding anglerfish peptide. Tilapia somatostatin-14 is identical to mammalian somatostatin but the [Tyr7, Gly10] somatostatin-containing peptide derived from prosomatostatin II contains the additional substitution (Phe11-->Leu) compared with the corresponding peptide from other teleosts.

Isolation of three antibacterial peptides from pig intestine: gastric inhibitory polypeptide (7-42), diazepam-binding inhibitor (32-86) and a novel factor, peptide 3910.

Two antibacterial peptides, cecropin P1 and PR-39 (39-residue proline/arginine-rich peptide), from the upper part of pig small intestine have previously been isolated and characterized. We have now continued our search for antibacterial peptides in different side fractions generated during the isolation of intestinal hormones. Starting from one such fraction and monitoring activity against Bacillus megaterium, we isolated three homogeneous peptides by three consecutive chromatographic steps. Amino acid sequence analysis in combination with mass spectrometry identified two of the peptides as gastric inhibitory polypeptide (7-42) [GIP(7-42)] and diazepam-binding inhibitor (32-86) [DBI(32-86)], derived from factors already known. However, intact GIP and DBI have hardly any antibacterial activity by themselves. The third peptide constitutes a previously unknown structure, designated as peptide 3910 from its molecular mass. All three peptides showed good activity against B. megaterium. In addition, GIP (7-42) showed some activity against Streptococcus pyogenes and an Escherichia coli mutant with a defect in its outer membrane.

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.

Isolation and bioactivity of putative cholecystokinin-releasing peptide from rat small intestinal mucosa.

Pancreatic exocrine secretion in the conscious rat is regulated by proteases in the intestine secreted by the pancreas, and cholecystokinin (CCK) is known to be involved in the mechanism. The authors proposed that the release of CCK was regulated by a CCK-releasing factor secreted into the intestinal lumen from the proximal intestine. We isolated and partially purified a CCK-releasing factor from rat small intestine by gel filtration and high performance liquid chromatography. The partially purified CCK-releasing factor increased pancreatic exocrine secretions and plasma CCK concentrations in conscious rats and this activity was abolished after the incubation with trypsin. The bioactivity of the partially purified CCK-releasing factor was confirmed.

Primary structure of frog PYY: implications for the molecular evolution of the pancreatic polypeptide family.

A peptide belonging to the pancreatic polypeptide (PP) family was isolated in pure form from the intestine of the European green frog (Rana ridibunda). The primary structure of the peptide was established as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu10-Asp-Ala- Ser-Pro-Glu-Glu-Met-Thr-Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile- Asn-Leu30-Val - Thr-Arg-Gln-Arg-Tyr-NH2. This amino acid sequence shows moderate structural similarity to human PYY (75% identity) but stronger similarity to the PP family peptides isolated from the pancreas of the salmon (86%) and dogfish (83%). The data suggest that the two putative duplications of an ancestral PP family gene that have given rise to PP, PYY and NPY in mammals had already taken place by the time of the appearance of the amphibia. In fish, however, only a single duplication has occurred, giving rise to NPY in nervous tissue and a PYY-related peptide in both pancreas and gut.

Identification, characterization and release of GRP gene-associated peptides from the normal porcine and human gastro-intestinal tract.

Using a radioimmunoassay directed towards human proGRP (42-53) on acetic acid extracts, immunoreactivity was measured throughout the porcine GI-tract in concentrations that were parallel to those of GRP (gastrin-releasing peptide or 'mammalian bombesin'). Gel filtration and HPLC studies of human and porcine tissue extracts revealed that the immunoreactivity was mainly due to a peptide with a molecular size of 8-9 kDa. The peptide did not contain the GRP sequence, making it a major fragment of the GRP C-flanking part of proGRP. Furthermore, a peptide of similar size with proGRP (42-53) immunoreactivity was released from isolated, perfused preparations of porcine antral and non-antral stomach and pancreas in parallel with GRP in response to electrical stimulation of the vagus nerves. Our results suggest that a processing of preproGRP occurs in normal, adult human and porcine tissues, that is similar to that previously demonstrated in small cell lung carcinomas and human fetal lungs. The finding that the immunoreactive proGRP fragment is released from the tissues upon appropriate stimulation raises the question of a possible physiological role for proGRP products other than GRP.

Identification of the 14 kDa bile acid transport protein of rat ileal cytosol as gastrotropin.

The 14 kDa bile acid binding protein of rat ileal cytosol (I-BABP), previously shown to be the major intracellular transporter of bile acids in enterocytes, was purified by affinity chromatography and gel electrophoresis. Enzymatic digestion of I-BABP which had been electroblotted to nitrocellulose led to the recovery and sequence analysis of four peptides representing 47 residues of sequence (approximately 35% of the full sequence). All the peptide sequences displayed high levels of identity (greater than 60%) and homology (greater than 80%) to the sequences of porcine and canine gastrotropin. This high level of homology together with other features of I-BABP identify it as rat gastrotropin, establishing gastrotropin as the major intracellular bile acid carrier of rat enterocytes.

Possible role of rat fatty acid-binding proteins in the intestine as carriers of phenol and phthalate derivatives.

Fatty acid-binding proteins of hepatic and intestinal type and gastrotropin-like protein (GTLP) were purified from rat intestinal cytosol by Sephadex G-75 gel filtration and DEAE-cellulose, CM-cellulose, and hydroxylapatite chromatographies. In addition to fatty acids, butylated hydroxytoluene (BHT), phthalate dibutyl, and di(2-ethylhexyl) esters (DBP and DEHP) were identified by gas liquid chromatography and mass spectrometry as endogenous ligands from the extract of either fatty acid-binding protein superfamily. These protein families in the intestine may have an important role as carriers in the initial step of arresting these exogenous pollutants.

Structural characterization of canine PYY.

PYY was purified from canine colonic mucosa by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence, amino acid and mass spectral analyses of the purified peptide and its tryptic fragments were consistent with the structure: YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY-amide. Canine PYY(1-36) has the identical sequence as porcine and rat PYY but differs from human PYY at position 3, with Ala instead of Ile, and position 18, with Ser instead of Asn. A smaller form, PYY(3-36), was also purified and characterized. It may differ in its biological activity from the intact peptide and could act as a partial antagonist or agonist of PYY(1-36).

Isolation and characterization of a 60-residue intestinal peptide structurally related to the pancreatic secretory type of trypsin inhibitor: influence on insulin secretion.

We have isolated from pig intestine a 60-residue polypeptide initially identified by its inhibition of glucose-induced insulin secretion from perfused pancreas. The amino acid sequence of this porcine polypeptide was determined and found to be markedly similar to that of the pancreatic secretory trypsin inhibitor (41% residue identities). Furthermore, the disulfide arrangements of these two proteins appear identical, suggesting related overall conformations. However, the polypeptide, now named PEC-60 (peptide with N-terminal glutamic acid, C-terminal cysteine, and a total of 60 residues), was found not to inhibit trypsin. The amino acid sequence is also similar to that of a peptide recently isolated from rat bile/pancreatic juice which stimulates the release of cholecystokinin. The biological role of PEC-60 is not known, but the effect on insulin secretion and the homologies observed suggest important biological activities and interesting structural relationships.

Complete sequences of glucagon-like peptide-1 from human and pig small intestine.

In the small intestine, proglucagon is processed into the previously characterized peptide "glicentin" (proglucagon (PG) 1-69) and two smaller peptides showing about 50% homology with glucagon: glucagon-like peptide-1 and -2. It was assumed that the sites of post-translational cleavage in the small intestine of the proglucagon precursor were determined by pairs of basic amino acid residues flanking the two peptides. Earlier studies have shown that synthetic glucagon-like peptide-1 (GLP-1) synthesized according to the proposed structure (proglucagon 71-108 or because residue 108 is Gly, 72-107 amide) had no physiological effects, whereas a truncated from of GLP-1, corresponding to proglucagon 78-107 amide, strongly stimulated insulin secretion and depressed glucagon secretion. To determine the amino acid sequence of the naturally occurring peptide we isolated GLP-1 from human small intestine by hydrophobic, gel permeation, and reverse-phase high performance liquid chromatography. By analysis of composition and sequence it was determined that the peptide corresponded to PG 78-107. By mass spectrometry the molecular mass was determined to be 3295, corresponding to PG 78-107 amide. Furthermore, mass spectrometry of the methyl-esterified peptide showed an increase in mass compatible with the presence of alpha-carboxyl amidation. Thus, the gut-derived insulinotrophic hormone GLP-1 is shown to be PG 78-107 amide.

Pig gastric (H+ + K+)-ATPase. Lys-497 conserved in cation transporting ATPases is modified with pyridoxal 5'-phosphate.

Pig gastric (H+ + K+)-ATPase can be covalently modified with pyridoxal 5'-phosphate (PLP) (about 1 mol/mol enzyme), and this modification is not observed in the presence of ATP, suggesting that PLP binds to a specific Lys residue in the ATP binding site or the region in its vicinity (Maeda, M., Tagaya, M., and Futai, M. (1988) J. Biol. Chem. 263, 3652-3656). The peptides labeled with radioactive PLP could be released from the gastric membrane vesicles quantitatively by chymotrypsin treatment, and two peptides were purified by high performance liquid chromatographies. These peptides were not obtained from vesicles incubated with PLP in the presence of ATP. The sequences of the two peptides were NH2-Asn-Ser-Thr-Asn-Lys-Phe-COOH and NH2-Ser-Thr-Asn-Lys-Phe-COOH, exactly corresponding to residues 493-498 and 494-498, respectively, of pig gastric (H+ + K+)-ATPase sequenced recently (Maeda, M., Ishizaki, J., and Futai, M. (1988) Biochem. Biophys. Res. Commun. 157, 203-209). Lys-497 was concluded to be the binding site of PLP, as pyridoxyl-Lys was identified at the corresponding position. This Lys residue is conserved in (Na+ + K+)- and Ca2+-ATPases. The possible amino acid residues in the catalytic site of gastric (H+ + K+)-ATPase are discussed.

Isolation and primary structure of human peptide YY.

The isolation, primary structure and chemical synthesis of human peptide YY (PYY) are described. The peptide was purified from human colonic extracts using a chemical method which detected the C-terminal tyrosine amide structure of PYY. Human PYY consists of 36 amino acid residues and the complete amino acid sequence is: Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu- Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu- Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2. The differences between the structures of porcine and human PYY are at positions 3 (Ala/Ile replacement) and 18 (Ser/Asn). Synthetic human PYY prepared using a solid-phase synthetic technique was found to be structurally identical to the natural peptide.