Rainbow trout (Oncorhynchus mykiss) neuropeptide Y.

Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Pro-Glu-Glu-Leu-Ala-Lys-Tyr-Tyr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchus kisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells.     

Isolation and primary structure of an amphibian neurotensin.

Using a radioimmunoassay system employing an antiserum which recognises the common C-terminal tripeptide (YIL) of neurotensin (NT) and neuromedin N (NN), immunoreactivity was identified in extracts of brain (65.8 pmol/g), small intestine (44.2 pmol/g) and rectum (13.2 pmol/g) of the European common frog (Rana temporaria). No immunoreactivity was detected in extracts of stomach and skin. Reverse-phase HPLC analysis of each tissue extract resolved a single immunoreactive peptide with identical retention time in each case. The immunoreactive peptide was isolated by reverse-phase HPLC from brain extracts and an N-terminal pyroglutamyl residue was successfully removed enzymatically. The molecular mass of des(pyroglutamyl) frog NT, determined by plasma desorption mass spectroscopy, was 1440 Da. The primary structure of this peptide was determined by gas-phase sequencing and the calculated molecular mass, 1440.7 Da, was in close agreement with that derived by mass spectroscopy. The full primary structure of frog NT was established as: QSHISKARRPYIL. When compared with bovine NT, frog NT exhibits five amino acid substitutions in the N-terminal region, whereas the C-terminal hexapeptide sequence (RRPYIL), which mediates the classical biological effects of NT, is completely conserved. Amphibia thus possess a tridecapeptide NT which is analogous to that of higher vertebrates and considerable constraints on the primary structure of the C-terminal biologically-active core have existed for a vast evolutionary time span.     

Isolation and characterization of neuropeptide Y from porcine intestine

The isolation and primary structure of intestinal neuropeptide Y (NPY) is described. The peptide was purified from porcine intestinal extracts using a chemical assay and radioimmunoassay for NPY. The amino acid sequence of this peptide is: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala- Arg-Tyr-Tyr- Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. This the structure of intestinal NPY is identical to the NPY of brain origin.     

Localization and identification of Neuropeptide Y (NPY)-like immunoreactivity in the frog brain

The distribution of neuropeptide Y (NPY) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. NPY-like containing perikarya were localized in the infundibulum, mainly in the ventral and dorsal nuclei of the infundibulum, in the preoptic nucleus, in the posterocentral nucleus of the thalamus, in the anteroventral nucleus of the mesencephalic tegmentum, in the part posterior to the torus semicircularis, and in the mesencephalic cerebellar nucleus. Numerous perikarya were also distributed in all cerebral cortex. Important tracts of immunoreactive fibers were found in the infundibulum, in the preoptic area, in the lateral amygdala, in the habenular region, and in the tectum. The cerebral cortex was also densely innervated by NPY-like immunoreactive fibers. A rich network of fibers was observed in the median eminence coursing towards the pituitary stalk. Scattered fibers were found in all other parts of the brain except in the cerebellum, the nucleus isthmi and the torus semicircularis, where no immunoreactivity could be detected. NPY-immunoreactive fibers were observed at all levels of the spinal cord, with particularly distinct plexus around the ependymal canal and in the distal region of the dorsal horn. At the electron microscope level, NPY containing perikarya and fibers were visualized in the ventral nuclei of the infundibulum, using the peroxidase-antiperoxidase and the immunogold techniques. NPY-like material was stored in dense core vesicles of 100 nm in diameter. A sensitive and specific radioimmunoassay was developed. The detection limit of the assay was 20 fmole/tube. The standard curves of synthetic NPY and the dilution curves for acetic acid extracts of cerebral cortex, infundibulum, preoptic region, and mesencephalon plus thalamus were strictly parallel. The NPY concentrations measured in these regions were (pmole/mg proteins) 163±8, 233±16, 151±12 and 60±13, respectively. NPY was not detectable in cerebellar extracts. After Sephadex G-50 gel filtration of acetic acid extracts from whole frog brain, NPY-like immunoreactivity eluted in a single peak. Reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay were used to characterize NPY-like peptides in the frog brain. HPLC analysis revealed that infundibulum, preoptic area and telencephalon extracts contained a major peptide bearing NPY-like immunoreactivity. The retention times of frog NPY and synthetic porcine NPY were markedly different. HPLC analysis revealed also the existence, in brain extracts, of several other minor components cross-reacting with NPY antibodies. These results provide the first evidence for the presence of NPY in the brain of a non-mammalian chordate and indicate that the structure of NPY is preserved among the vertebrate phylum. The abundance of NPY producing neurons in the hypothalamus and telencephalon suggests that this peptide may play both neuroendocrine and neurotransmitter functions in amphibians.     

The primary structure of peptide Y (PY) of the spiny dogfish, Squalus acanthias: immunocytochemical localisation and isolation from the pancreas.

1. Endocrine cells within islets, exocrine parenchyma and ductal epithelium in the pancreas of the spiny dogfish, Squalus acanthias, were immunostained with an antiserum to the C-terminal region of mammalian neuropeptide Y (NPY). 2. Radioimmunoassay of pancreatic extracts with the same antiserum detected immunoreactivity in the dorsal lobe (338 pmol/g) and ventral lobe (433 pmol/g). Reverse phase HPLC analysis of both extracts resolved a single immunoreactive peptide. 3. The primary structure of the isolated peptide was established as: YPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY.NH2. 4. Peptide Y (PY) from Squalus acanthias is identical in primary structure to an NPY-related peptide isolated from the pancreas of Scyliorhinus canicula and has a 31/36 residue homology with porcine NPY. The 5 substitutions are highly-conservative.     

The complete primary structure of pancreatic polypeptide from the European common frog, Rana temporaria

Using an antiserum directed against the highly-conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP), numerous immunoreactive endocrine cells were identified within the pancreas of the European common frog, R. temporaria. An acidified ethanolic extract of pancreatic tissue (0.859 g, n = 35) contained 26.2 nmol equivalents/g of tissue. Gel permeation chromatography of the extract resolved a single peak of immunoreactivity co-eluting with synthetic bovine PP standard. Reverse phase HPLC of this material resolved a single peak of immunoreactivity which was purified to homogeneity following chromatography on a semipreparative C-18 column and an analytical C-8 column. Plasma desorption mass spectrometry (PDMS) of the purified peptide resolved a single component with a molecular mass of 4240.9 Da. Direct gas phase sequencing established the sequence of the first 26 residues. Following incubation of the peptide with endopeptidase Asp-N and direct application of the digest to the sequencer, the entire primary structure of the peptide was established as: APSEPHHPGDQATQDQLAQYYSDLYQYITFVTRPRF. The derived molecular mass of this peptide, incorporating a C-terminal amide, was 4240.6 Da which is entirely consistent with that obtained by PDMS.     

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.     

PD-sauvagine: a novel sauvagine/corticotropin releasing factor analogue from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor

Sauvagine is a potent and broad-spectrum biologically active peptide of 40 amino acid residues originally isolated from the skin of the South American frog, Phyllomedusa sauvagei. Since its discovery, no additional sauvagine structures have been reported. Following the discovery of sauvagine, peptides with similar primary structures/activities were identified in mammalian brain [corticotropin-releasing factor (CRF) and urocortin]. Here, we report the identification of a second sauvagine from the Mexican giant leaf frog, Pachymedusa dacnicolor, which displays primary structural features of both sauvagine and CRF. A cDNA encoding the peptide precursor was "shotgun" cloned from a cDNA library constructed from lyophilised skin secretion by 3'- and 5'-RACE reactions. From this, the primary structure of a 38-mer peptide was deduced and this was located in reverse phase HPLC fractions of skin secretion and both its mass and structure were confirmed by mass spectrometry. The biological activities of synthetic replicates of PD-sauvagine and sauvagine were compared using two different mammalian smooth muscle preparations and the novel peptide was found to be more potent in both. Bioinformatic analyses of PD-sauvagine revealed that it shared different regional sequence identities with both sauvagine and CRF.     

An atypical member of the brevinin-1 family of antimicrobial peptides isolated from the skin of the European frog Rana dalmatina

A single peptide with antimicrobial activity was extracted from the skin of the European agile frog (R. dalmatina). The primary structure of this 17 amino-acid-residue peptide (ILPLLLGKVVCAITKKC) does not immediately suggest membership of any of the previously described families of antimicrobial peptides from ranid frogs. However, if it is assumed that the peptide has undergone several residue deletions during the course of speciation, it shows sequence similarity with peptides belonging to the widely distributed brevinin-1 family, particularly those isolated from the related species Rana temporaria. The minimum inhibitory concentration of the peptide, termed brevinin-1 Da, against the Gram-positive bacterium Staphylococcus aureus was 7 microM and against the Gram-negative bacterium Escherichia coli was 30 microM.     

Purification and structure of neurostatin, an inhibitor of astrocyte division of mammalian brain

Neurostatin was originally described as an inhibitor of astroblast and astrocytoma division present in rat brain extracts and immunologically related to the sugar moiety of epidermal growth factor receptor and to blood group antigens. It was purified recently from mammalian brain extracts and characterized as a glycosphingolipid, but its precise structure remained unknown. Neurostatin has now been purified to apparent homogeneity from ganglioside extracts of rat, bovine, and porcine brain. It is cytostatic for astroblasts, C6 glioma cells, and various human astrocytomas grades III and IV, with IC(50) values ranging from 250 to 450 nM, but does not affect the division of primary or transformed fibroblasts up to concentrations >4 microM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of purified pig neurostatin showed a molecular ion of 1, 905 Da and ions of 1,863 and 1,934 Da, compatible with a disialoganglioside. Mono- and bidimensional NMR spectra, together with biochemical studies, suggest that neurostatin may be the 9-O-monoacetyl ester of GD1b.     

Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog,Agalychnis callidryas

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.     

Neuropeptide Y in the intermediate lobe of the frog pituitary acts as an alpha-MSH-release inhibiting factor

The presence of neuropeptide tyrosine (NPY) in the intermediate lobe of the frog pituitary was demonstrated using indirect immunofluorescence, the immunogold technique and a specific radioimmunoassay combined with high pressure liquid chromatography (HPLC). A high density of NPY-containing fibers, was found among the parenchymal cells of the intermediate lobe. These fibers originated from the ventral infundibular nucleus, travelled via the median eminence to the pars intermedia. At the electron microscopic level, NPY-like material was found exclusively in nerve fibers where the product of the immunoreaction was associated to dense-core vesicles. High concentrations of NPY-like peptide were found in neurointermediate lobe extracts. After Sephadex G-50 gel filtration the major peak of immunoreactive material appeared to co-elute with synthetic porcine NPY. Conversely, HPLC analysis revealed that the NPY-like peptide of the frog pituitary had a retention time shorter than the porcine NPY. The localization of NPY-like material in the pars intermedia suggested a possible role of NPY in the regulation of melanotropic cell secretion. In fact, graded concentrations of synthetic NPY induced a dose-dependent inhibition of alpha-melanotropin (alpha-MSH) release in vitro. The lack of effect of a dopaminergic antagonist on NPY-induced alpha-MSH release inhibition demonstrated that the local dopaminergic system could not account for the NPY action. These results indicate that NPY located in the hypothalamo-hypophyseal system of the frog may act as a melanotropin-release inhibiting factor.     

Characterization of aPY-like peptides in anglerfish brain using a novel radioimmunoassay for aPY-Gly

Anglerfish peptide YG (aPY) was isolated from pancreatic islets of the anglerfish. Subsequent immunohistochemical and biochemical analyses demonstrated that anglerfish islet cells synthesize aPY. We have now developed and characterized a radioimmunoassay (RIA) for aPY and have examined extracts of anglerfish brain for aPY-like peptides. Brain extracts were subjected to gel filtration and high performance liquid chromatography (HPLC). Fractions from HPLC eluates were analyzed in the aPY RIA and also in a neuropeptide Y (NPY) RIA. A single peak of aPY-like immunoreactivity eluted from HPLC columns. The elution position of this aPY-like peptide coincided exactly with the aPY-Gly marker under several gradient conditions. Results from the NPY RIA confirmed the presence of several molecular forms of NPY-like immunoreactive peptides in the anglerfish brain. These results demonstrate the utility of the newly developed aPY RIA for studies of anglerfish brain peptides and extend our previous immunohistochemical demonstration of aPY-like staining in the anglerfish brain.     

Radioimmunoassay of neuropeptide Y

The development of a radioimmunoassay to the newly isolated peptide, neuropeptide Y is described. Four separate antisera have been developed using different immunisation schedules. Two of these antisera (YNI and YNIO) are directed to the C-terminal region of the peptide and cross-react with the related peptide PYY, whereas YN7 is specific being directed to the N-terminal region of NPY, YN6 is similarly specific for NPY, but is unable to bind the available fragments. These four antisera provide similar results for determination of NPY immunoreactivity within porcine brain extracts, however YN6 consistently undervalues all extracts from the other species examined (human, rat, guinea pig, cat and mouse). Chromatographic analysis by means of reverse phase high pressure liquid chromatography (HPLC) shows that NPY immunoreactivity of human extracts elutes in an earlier position than the porcine standard. It seems likely therefore that human and porcine NPY differ in their amino acid sequences.     

Isolation and sequence of rat peptide YY and neuropeptide Y

Rat peptide YY and rat neuropeptide Y have been isolated in parallel from colon and brain extracts respectively, using salt fractionation, gel filtration chromatography, cation-exchange HPLC, and reverse phase phenyl-silica HPLC. Immunoreactivity was identified using a combination of 3 NPY immunoassays which exhibit differing cross-reactivities for PYY (90%, less than 0.01% and 30% respectively). The yield at the final purification step was 1.2 nmol rPYY and 0.5 nmol rNPY. Half of each purified peptide was subjected to complete microsequence analysis. This showed that while rat NPY was structurally identical to human NPY, the sequence of PYY from rat colon was the same as porcine PYY isolated from extracts of duodenum.     

Solubilization of the Neuropeptide Y Receptor from Rat Brain Membranes

The neuropeptide Y (NPY) receptor was solubilized from rat brain membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The binding of 125I-NPY to CHAPS extracts was protein, time, and temperature dependent. Unlabeled NPY and the related peptides peptide YY (PYY) and pancreatic polypeptide inhibited 125I-NPY binding to solubilized receptors with relative potencies similar to those seen with membrane-bound receptors: NPY greater than PYY much greater than pancreatic polypeptide. Scatchard analysis of equilibrium binding data showed the CHAPS extracts to contain a single population of binding sites with a KD of 3.6 +/- 0.4 nM (mean +/- SEM) and a Bmax of 5.0 +/- 0.2 pmol/mg of protein. In addition the 125I-NPY binding to the soluble receptor was not inhibited by guanosine-5'-O-(3-thiotriphosphate), in contrast to the GTP sensitivity displayed by the membrane-bound receptor. Gel filtration chromatography using Sepharose 6B revealed a single peak of binding activity corresponding to a Mr of approximately 67,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after chemical cross-linking revealed a single band at Mr 62,000. After solubilization and gel chromatography a 50- to 100-fold purification of the NPY receptor was obtained.     

Direct analysis of the kinetic profiles of organophosphate-acetylcholinesterase adducts by MALDI-TOF mass spectrometry

A sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure has been established for the detection and quantitation of acetylcholinesterase (AChE) inhibition by organophosphate (OP) compounds. Tryptic digests of purified recombinant mouse AChE (mAChE) were fractionally inhibited by paraoxon to form diethyl phosphoryl enzyme. The tryptic peptide of mAChE that contains the active center serine residue resolves to a molecular mass of 4331.0 Da. Phosphorylation of the enzyme by paraoxon results in covalent modification of the active center serine and a corresponding increase in molecular mass of the tryptic peptide by 136 Da. The relative abundance of AChE peptides containing a modified active center serine strongly correlates with the fractional inhibition of the enzyme, achieving a detection range of phosphorylated to nonphosphorylated enzyme of 5-95%. Modifications of AChE by OP compounds resulting in dimethyl, diethyl, and diisopropyl phosphoryl adducts have been monitored with subpicomole amounts of enzyme. The individual phosphorylated adducts of AChE that result from loss of one alkyl group from the inhibited enzyme (the aging reaction) and the reappearance of unmodified AChE (spontaneous reactivation) have been resolved by the kinetic profiles and relative abundance of species. Further, the tryptic peptide containing the active center serine of AChE, isolated from mouse brain by anion-exchange and affinity chromatography, has been monitored by mass spectrometry. Native brain AChE, purified from mice treated with sublethal doses of metrifonate, has demonstrated that enzyme modifications resulting from OP exposure can be detected in a single mouse brain. For dimethyl phosphorylated AChE, OP exposure has been monitored by the ratio of tryptic peptide peaks that correspond to unmodified (uninhibited and/or reactivated), inhibited, and aged enzyme.     

Sauvatide - A novel amidated myotropic decapeptide from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei

Here we describe the structural and functional characterization of a novel myotropic peptide, sauvatide, from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei. Sauvatide is a C-terminally amidated decapeptide with the following primary structure - LRPAILVRTKamide - monoisotopic mass 1164.77 Da, which was found to contract the smooth muscle of rat urinary bladder with an EC₅₀ of 2.2 nM. The sauvatide precursor, deduced from cloned skin cDNA, consists of 62 amino acid residues with a single copy of sauvatide located near the C-terminus. The mature peptide is generated from the precursor by cleavage at a classical -KR-cleavage site located proximal to the N-terminus and by removal of a -GKGK sequence at the C-terminus, the first glycyl residue acting as amide donor. Amphibian skin secretions thus continue to be a source of novel and potent biologically active peptides acting through functional targets in mammalian tissues.     

Anglerfish islets contain NPY immunoreactive nerves and produce the NPY analog aPY

It has recently been demonstrated that aPY, a peptide which has significant homology with neuropeptide Y (NPY) is present in extracts of anglerfish islets. The purpose of this study was to determine whether cells or nerves which contain NPY-like immunoreactivity could be identified in anglerfish islet tissue and whether aPY is synthesized by this tissue. Antisera against bovine pancreatic polypeptide (BPP), NPY and the 200 kd neurofilament polypeptide were used for immunohistochemical analysis of islets. Identical cells were stained by both the NPY and BPP antisera. The NPY and 200 kd neurofilament antisera also labeled nerve fibers in the tissue which were not stained with the BPP antiserum. The nature of the NPY-like peptide synthesized in islet cells was determined by subjecting differentially radioactively labeled Mr 2,500-8,000 peptides from islet extracts to reverse phase HPLC. Labeled aPY was unequivocally identified in the extracts and was labeled appropriately (as predicted from its sequence) with 13 different radioactive amino acids. These results demonstrate that one form of NPY-like peptide synthesized in anglerfish islets is aPY. The form of NPY-like peptide which was immunolocalized in nerves remains to be determined.     

Synthesis and biological properties of 4-norleucine-neuropeptide Y; secondary structure of neuropeptide Y

Neuropeptide Y (NPY) is a 36 amino acid peptide amide isolated from porcine brain. The NPY analog, 4-norleucine-NPY was synthesized by a solid-phase method and purified to homogeneity in 20% yield by reverse-phase chromatography. Investigation of the biological properties indicated that the analog is an agonist of NPY. Secondary structural analyses revealed that NPY and the analog exhibited predominantly alpha-helical and beta-sheet structures, respectively; however, experiments in trifluoroethanol indicated that the analog has the potential of assuming an alpha-helical structure. Based on circular dichroism (CD), Raman spectroscopy and Chou-Fasman analyses, a model has been proposed for the secondary structure of NPY.