J.Michael Conlon

Scyliorhinin I and II: two novel tachykinins from dogfish gut

Two peptides with tachykinin‐like ability to contract longitudinal muscle from the guinea pig ileum were isolated from the intestine of the common dogfish, Scyliorhinus caniculus. The amino acid sequence of scyliorhinin I was established as Ala‐Lys‐Phe‐Asp‐Lys‐Phe‐Tyr‐Gly‐Leu‐Met‐NH2 and this peptide cross‐reacted with antisera directed against the C‐terminal region fo substance P. The amino acid sequence of scyliorhinin II was established as Ser‐Pro‐Ser‐Asn‐Ser‐Lys‐Cys‐Pro‐Asp‐Gly‐Pro‐Asp‐Cys‐Phe‐val‐Gly‐Leu‐Met‐NH2 and this peptide cross‐reacted with antisera directed against the C‐terminal region of neurokinin A. The mammalian peptides substance P and neurokinin A were absent from the dogfish intestinal tissue.

Somatostatin-related and glucagon-related peptides with unusual structural features from the European eel (Anguilla anguilla)

Peptides derived from prosomatostatins I and II and from two distinct proglucagons have been isolated from the pancreas of a teleost fish, the European eel (Anguilla anguilla). The product of prosomatostatin I processing, somatostatin-14, is identical to mammalian somatostatin-14. A 25-amino-acid-residue peptide (Ser-Val-Asp-Asn-Gln5-Gln-Gly-Arg-Glu-Arg10-Lys-Ala-Gly-Cys- Lys15-Asn-Phe-Tyr- Trp-Lys20-Gly-Pro-Thr-Ser-Cys25) is derived from prosomatostatin II. Compared with the corresponding peptides from other teleost fish, the eel somatostatin-25 contains the unusual substitution Pro for Phe at position 22. This peptide was also isolated in a form containing a hydroxylsyl residue at position 20. A 29-amino-acid-residue eel glucagon contains four substitutions relative to human glucagon Asn for Ser8, Glu for Asp15, Thr for Ser16, and Ser for Thr29). In common with mammalian and avian glucagons but unlike most other fish glucagons, the eel peptide possesses a glutamine residue at position 3. A peptide derived from a second proglucagon comprises 36 amino acid residues. A 7-residue C-terminal extension to the glucagon sequence shows structural similarity to the corresponding extension in ratfish (Hydrolagus colliei) glucagon and mammalian oxyntomodulin.

Calcitonin gene-related peptides relax guinea pig and rat gastric smooth muscle.

Rat and human alpha- and beta-calcitonin gene-related peptide (CGRP), in the concentration range 1-100 nM, produced sustained relaxations of longitudinal muscle from the rat fundus and guinea pig gastric corpus. The peptides were equipotent and equally effective. Tetrodotoxin, adrenoceptor and purine receptor antagonists, somatostatin, apamin and Tyr-rat alpha-CGRP-(28-37) peptide did not modify the action of the CGRP peptides. The CGRP-induced responses were inhibited by verapamil and potentiated by Bay K-8644. Incubation of the tissues with indomethacin markedly reduced the magnitude of the CGRP- and adrenaline-induced relaxations, but their responsiveness was restored by addition of prostaglandins E1, E2 and F2 alpha in concentrations that alone did not affect the motility of the indomethacin-treated strips. It is suggested that an inhibitory receptor for CGRP on gastric smooth muscle cells is linked to calcium channels and may be activated or sensitized by endogenous prostaglandins.

Multiple molecular forms of insulin and glucagon-like peptide from the pacific ratfish (Hydrolagus colliei)

The primary structure of an insulin isolated from the pancreas of the holocephalan fish, Hydrolagus colliei (Pacific ratfish), has been established as A-chain: GIVEQCCHNTCSLANLEGYCN B-chain: VPTQRLCGSHLVDALYFVCGERGFFYSPKPIRELEPLL. Three further molecular forms of insulin were also isolated and shown to have the same A-chain but truncated B-chains of 31-, 36-, and 37-amino acid residues. It is proposed that all four insulins arise from a single proinsulin by proteolytic cleavages at different sites within the C-peptide region. The insulin with 38 amino acids in the B-chain was equipotent with human insulin in inhibiting the binding of radiolabelled human insulin to rat fat cells but the maximum effect of ratfish insulin upon the transport of 3-O-methylglucose into the cells was only 65% of the maximum effect of human insulin. Two molecular forms of glucagon-like peptide were isolated from the ratfish pancreas. The primary structure of the more abundant peptide was established as HADGIYTSDVASLTDYLKSKRFVESLSNYNRKQND. The primary structure of the second peptide was the same except that it was extended from the C-terminus by the sequence RRM. It is probable, therefore, that both glucagon-like peptides also arise from a single proglucagon by different pathways of post-translational processing.

Primary structure of insulin and a truncated C-peptide from an elasmobranchian fish, Torpedo marmorata

Insulin has been isolated from the pancreas of Torpedo marmorata, an elasmobranchian fish, and shown to contain 21 amino acid residues in the A-chain and 30 residues in the B-chain. The sequence of insulin has been strongly conserved within the class Elasmobranchii with only one substitution and one deletion in the A chain and one substitution in the B-chain compared with insulin from the spiny dogfish, Squalus acanthias. A second peptide, present in the pancreatic extracts in approximately equimolar concentration with insulin, was identified as a heptadecapeptide. The sequence of this peptide shows homology to the N-terminal region of anglerfish (Lophius americanus) C-peptide at six of 17 sites. The isolation of a truncated C-peptide suggests either that the sequence encoding the COOH-terminal region of T. marmorata C-peptide has been deleted from the preproinsulin gene or that a larger C-peptide has undergone a proteolytic cleavage in the central portion of the molecule during packaging in the secretory granules of the B cell.

Primary structure of insulin and glucagon from the flounder (Platichthys flesus)

Insulin and glucagon have been isolated from the Brockmann bodies of the flounder, a teleostean fish, and their primary structures established by automated Edman degradation. The A-chain of flounder insulin shows strong homology to the A-chains from the coho salmon (Oncorhynchus kisutch; 100%) and the anglerfish (Lophius americanus; 95%) but homologies in the B-chain region are weaker (salmon 79%, anglerfish 83%). Flounder insulin B-chain contains the novel sequence Val-Val-Pro-Pro at the NH2 terminus and the highly conserved seryl residue at position 10 (B 9 in mammals) is replaced by an alanyl residue. Flounder glucagon is identical to anglerfish glucagon II but shows four amino acid substitutions compared with salmon glucagon.

Primary structure of glucagon from the gut of the common dogfish (Scyliorhinus canicula)

The primary structure of glucagon isolated from the intestine of the common dogfish, Scyliorhinus canicula, was established as 5 10 15 20 25 H S E G T F T S D Y S K Y M D N R R A K D F V Q W L M N T. The peptide shows four substitutions compared with human glucagon: Glu‐3 for Gln, Met‐14 for Leu, Asn‐16 for Ser and Lys‐20 for Gln. Glucagon represented the predominant molecular form of the glucagon‐like immunoreactivity in the dogfish gut extracts demonstrating that the pathway of posttranslational processing of proglucagon in the gut of this fish differs markedly from the pathway in the mammalian gut.

Isolation of the tachykinin, des[Ser1Pro2]scyliorhinin II from the intestine of the ray, Torpedo marmorata.

A peptide with neurokinin A-like immunoreactivity was isolated from an extract of the intestine of an elasmobranch fish, Torpedo marmorata. The primary structure of the peptide was established as Ser-Asn-Ser-Lys-Cys-Pro-Asp-Gly-Pro-Asp-Cys-Phe-Val-Gly-Leu-Met.NH2. This amino acid sequence is identical to that of residues (3-18) of scyliorhinin II previously isolated from the intestine of the common dogfish (Scyliorhinus canicula). The presence of the truncated peptide, lacking Ser-Pro, in the Torpedo gut suggests that scyliorhinin II may be a substrate for an enzyme with dipeptidylpeptidase IV-like specificity. The data support previous assertions that strong evolutionary pressure has acted within the elasmobranch subclass of chondrichthyean fish to conserve the structures of regulatory peptides.

[Ser7]neurotensin: isolation from guinea pig intestine

Using three antisera to neurotensin of defined regional specificity, a novel neurotensin has been identified in extracts of guinea pig brain and small intestine. The primary structure of the peptide was established as: pGlu Leu Tyr GIu Asn Lys Ser Arg Arg Pro Tyr He Leu. Guinea pig neurotensin differs from bovine neurotensin by substitution of a prolyl residue by a seryl residue at position 7. Synthetic [Ser7]neurotensin showed identical Chromatographie and immunochemical properties to guinea pig neurotensin. This difference in primary structure may account for some of the anomalous pharmacological effects of bovine neurotensin on guinea pig tissues.

Valosin: isolation and characterization of a novel peptide from porcine intestine

It does not show structural homology to known protein or nucleotide sequences. According to preliminary experiments, valosin possesses biological effects on the gastrointestinal tract of the dog.