Orcokinin: a novel myotropic peptide from the nervous system of the crayfish, Orconectes limosus.

A myotropic peptide, named orcokinin, was isolated from approximately 1200 abdominal nerve cords of the crayfish, Orconectes limosus. Its amino acid sequence was determined as follows: Asn-Phe-Asp-Glu-Ile-Asp-Arg-Ser-Gly-Phe-Gly-Phe-Asn. This structure was confirmed by synthesis. There is no sequence similarity to any known neuropeptide. Orcokinin exhibits high potency on the crayfish hindgut, enhancing both frequency and amplitude of spontaneous contractions. The threshold of biological activity in vitro was determined to be approximately 5 x 10(-11) M.

Structure-activity relationships of the crustacean myotropic neuropeptide orcokinin.

Orcokinin (OK, NFDEIDRSGFGFN) was recently identified from the crayfish, Orconectes limosus, as a potent hindgut-stimulating factor (14). To assess the importance of structural features of the peptide involved in effective ligand-receptor interactions, synthetic analogues of orcokinin were tested in the hindgut bioassay. Tests with N- and C-terminal-truncated analogues and the C-terminal-amidated analogue (OK-NH2) demonstrate that changes at the C-terminus interfere less with biological activity than changes at the N-terminus. Removal of more than one amino acid at the N-terminus resulted in a complete loss of activity, whereas the C-terminal deletion of three amino acids still produced an analogue with full intrinsic activity but with a drastic shift in threshold concentration of activity from 1 x 10(-10) to 1 x 10(-7) M. Deletion of four amino acids at the C-terminus resulted in a completely inactive analogue. The C-terminal hydroxyl group does not seem to be important because amidation (OK-NH2) resulted in almost no loss of activity. Replacing Arg7 with Ala produced an analogue almost equipotent to orcokinin. Replacement of Phe2 by Tyr resulted in considerable loss of activity. An important role of Phe2 is further suggested by the steep drop of activity after removal of this residue in the N-terminal-deleted analogues.

Two orcokinins and the novel octapeptide orcomyotropin in the hindgut of the crayfish Orconectes limosus: identified myostimulatory neuropeptides originating together in neurones of the terminal abdominal ganglion.

The tridecapeptides Asn(13)-orcokinin and Val(13)-orcokinin, two known members of the orcokinin neuropeptide family native to crustaceans, and a novel octapeptide, orcomyotropin, FDAFTTGFamide, have been identified from extracts of hindguts of the crayfish Orconectes limosus using an isolated hindgut contractility bioassay, high-performance liquid chromatography, microsequencing and mass spectrometry. All three peptides display strong inotropic actions on crayfish hindguts. Orcomyotropin showed higher potency than the two orcokinins. Threshold concentration was approximately 5 x 10(-12)mol l(-1)versus 10(-10)mol l(-1) for the two orcokinins. An approximately fivefold increase in contraction amplitude was observed with 10(-9)mol l(-1) orcomyotropin and 10(-7)mol l(-1) of the orcokinins. Asn(13)- and Val(13)-orcokinin did not differ significantly with regard to their biological effects. Semi-isolated crayfish hearts and locust oviducts did not respond to the three peptides. Immunocytochemistry using antisera against Asn(13)-orcokinin and orcomyotropin showed that these neuropeptides are co-localized in approximately 80-90 neurones of the terminal abdominal ganglion that have been shown to innervate the entire hindgut muscularis via the intestinal nerve. The neurones form elaborate terminal branches preferentially on longitudinal hindgut muscles. Orcomyotropin is a novel crustacean member of the GF-amide family of myotropic and/or allatotropic neuropeptides from annelids, molluscs and insects.