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The rabbit cerebellum has been shown to contain significant quantities of opioid receptors consisting of both mu- and kappa-subtypes. To determine the nature of the endogenous opioid ligands in this tissue, extracts from rabbit cerebellum were separated by various chromatography techniques and fractions were assayed initially for opioid peptides with a radioimmunoassay capable of detecting all peptides with an amino-terminal Tyr-Gly-Gly-Phe sequence. This sequence is common to all mammalian opioid peptides and is critical for recognition by all known opioid receptors. Each of the three immunoreactive opioid peptide peaks detected was purified to homogeneity and subjected to amino acid composition and sequence analysis. One peak was analyzed further by mass spectrometry. This identified the major opioid peptides in the cerebellum as [Met5]enkephalin, [Leu5]enkephalin, and heptapeptide [Met5]enkephalyl-Arg6-Phe7. The comprehensiveness of this initial detection scheme in identifying biologically active opioid peptides was substantiated through subsequent analysis. Using specific radioimmunoassays for representative opioid peptides of the three opioid systems currently known, no other peptides of either the proenkephalin, proopiomelanocortin, or prodynorphin series were detected in any appreciable amounts. Collectively, these results are consistent with the position that rabbit cerebellar opioids are derived from proenkephalin. However, given that no appreciable quantities of either [Met5]enkephalyl-Arg6-Arg7-Val8-NH2 (metorphamide) or [Met5]enkephalyl-Arg6-Gly7-Leu8 were detected suggests that rabbit proenkephalin may have a slightly altered sequence and/or is differentially processed relative to other mammalian species studied.

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Bovine pancreastatin, a 47 amino acid residue peptide, was isolated from the pancreas and the pituitary gland using a chemical method which detects its C-terminal glycine amide structure. The complete amino acid sequence of the pancreatic peptide is 74% homologous to that of porcine pancreastatin and is identical to bovine chromogranin A-(248-294), as deduced from its cDNA sequence. The sequence of the first 28 amino-terminal residues of the pituitary peptide was determined to be identical to the corresponding sequence of the pancreatic peptide. Since the pituitary peptide also contains the C-terminal glycine amide, it is therefore likely to be identical in structure to the pancreatic peptide. Thus, we conclude that bovine chromogranin A is the precursor of bovine pancreastatin. Synthetic bovine pancreastatin inhibited pancreatic exocrine secretion in a similar manner to porcine pancreastatin.

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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.

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Monoclonal antibodies were previously used to identify two glycoproteins, called fasciclin I and II (70 and 95 kDa, respectively), which are expressed on different subsets of axon fascicles in the grasshopper (Schistocerca americana) embryo. Here the monoclonal antibodies were used to purify these two membrane-associated glycoproteins for further characterization. Fasciclin II appears to be an integral membrane protein, whereas fasciclin I is an extrinsic membrane protein. The amino acid sequences of the amino terminus and fragments of both proteins were determined. Using synthetic oligonucleotide probes and antibody screening, we isolated genomic and cDNA clones. Partial DNA sequences of these clones indicate that they encode fasciclins I and II.

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In mammalian tissues the C-terminal amide structure has been found to occur only in neuroactive or hormonally-active peptides. About half known neuropeptide and peptide hormones have this unique chemical feature. Using a chemical detection method, a search for previously unknown peptides that possess the C-terminal amide structure in extracts of brain and intestine was carried out and a number of novel neuropeptides and hormonal peptides, designated neuropeptide Y, PHI, peptide YY, galanin and neuropeptide K were isolated. We recently performed a similar search in porcine pancreas and found a high concentration of a peptide having a glycine amide at its C-terminus. Here we report the isolation, primary structure and biological activity of this novel peptide. The 49-residue peptide strongly inhibits glucose-induced insulin release from the isolated perfused pancreas and was therefore named pancreastatin. It may be important in the regulation of insulin secretion and in the pathogenesis and treatment of diabetes mellitus.

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The large neurons of the mollusc Aplysia are useful for studying the biogenesis of neuropeptides in single cells. Neuron R14 in the abdominal ganglion synthesizes large quantities of a 10-kDa neuropeptide precursor. The amino acid sequence of this precursor has been defined by analysis of the nucleotide sequence of a cDNA clone. We labeled proteins in vivo by microinjection of radioactive amino acids into individual R14 neurons. The labeled peptides were fractionated by high performance liquid chromatography and subjected to Edman degradation, thus enabling us to determine post-translational processing sites. Cleavage of the signal sequence was observed and at two internal sites. Cleavage at these internal sites occurs at basic amino acids and results in three products, a 2.9-, a 4.9-, and a 1.4-kDa peptide. These studies of protein processing serve as a basis for further investigations of the biogenesis and physiological activities of the neuropeptides.

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Opioid-like immunoreactive material was extracted from the pituitary and brain of the Spiny Dogfish Shark Squalus acanthias. The immunoreactive material in the pituitary extracts was purified to apparent homogeneity by reverse phase high performance liquid chromatography and subsequently characterized by amino acid analysis, Edman degradation and fast atom bombardment mass spectrometry. The largest opioid-like peptide isolated contained 30 amino acids and showed 80 percent homology with salmon endorphin-II but less than 50 percent homology with human beta-endorphin. Three structural variants of this molecule were also characterized. These variants were shown to be shorter N-terminal fragments, two of which corresponded to cleavage products at the single basic residues arginine and lysine. Cleavage at a single lysine residue has not been reported for posttranslational processing of beta-endorphin in mammals and could represent a modification seen only in lower vertebrates. The remaining fragment corresponded to a loss of 3 residues from the C-terminus of the parent molecule. No alpha-N-acetylated peptides were detected. These results provide the first unequivocal confirmation of beta-endorphin in an elasmobranch and provide evidence of novel N-terminal variants of beta-endorphin.

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Using an antiserum directed at the COOH-terminus of tachykinins, we have examined postmortem tissue from two cases of metastatic ileal carcinoid for the presence of tachykinin-like immunoreactivity. The vast majority of the immunoreactive tachykinin-like material eluted from a Sephadex G-50 column as two peaks at positions corresponding to molecular weights of 1300 and 850. The 1300 dalton peak was resolved by reverse-phase-HPLC into two components which by Edman sequencing, amino acid analysis, and fast atom bombardment (FAB)-mass spectrometry criteria, were identified as substance P and substance K. The 850 dalton peak was also resolved on RP-HPLC into two peaks which were resistant to Edman degradation but from amino acid analysis and FAB-mass spectrometry criteria were identified as pyro-Glu-substance P 5-11 and oxidized pyro-Glu-substance P 5-11. In control experiments substance P 5-11 was converted to pyro-Glu-substance P 5-11 during the extraction procedure. Both tumors also contained a minor immunoreactive peak which eluted from a Sephadex G-50 sizing column at a position corresponding to a molecular weight of 4000 which probably represents neuropeptide K. These results suggest that beta-preprotachykinin is preferentially expressed in carcinoid tumors and that substance K may also play a role in the carcinoid syndrome.

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