Occurrence of analogues of the myotropic neuropeptide orcokinin in the shore crab, Carcinus maenas: evidence for a novel neuropeptide family

Abstract

By use of an enzyme immunoassay that was developed for the determination of orcokinin, a myotropic neuropeptide of the sequence NFDEIDRSGFGFN from the crayfish, Orconectes limosus, immunoreactive material was detected in extracts of thoracic ganglia from the shore crab, Carcinus maenas. Isolation of the immunoreactive material was achieved by the following steps: 1) prepurification by gel filtration, 2) immunoaffinity chromatography on an anti-orcokinin IgG protein-A sepharose column, and 3) reversed-phase HPLC. The HPLC profile after affinity purification revealed three main immunoreactive peptides that were rechromatographed. None of these peptides was identical to orcokinin in terms of retention time. Automated gas-phase sequencing revealed these peptides to be analogues of orcokinin differing in one amino acid residue. They were named [Ser9]-, [Ala13]- and [Val13]orcokinin (NFDEIDRSSFGFN, Mr 1549.3; NFDEIDRSGFGFA, Mr 1475.3; NFDEIDRSGFGFV, Mr 1503.9). Carboxypeptidase A treatment of the peptides indicated a free C-terminus. Complete characterization of the three peptides was achieved from approximately 230 thoracic ganglia of Carcinus maenas.