BINDING STUDIES OF NOVEL, NON-MAMMALIAN ENKEPHALINS, STRUCTURES PREDICTED FROM FROG AND LUNGFISH BRAIN cDNA SEQUENCES

Leu- and Met-enkephalin were the first endogenous opioid peptides identified in different mammalian species including the human. Comparative biochemical and bioinformatic evidence indicates that enkephalins are not limited to mammals. Various prodynorphin (PDYN) sequences in lower vertebrates revealed the presence of other enkephalin fingerprints in these precursor polypeptides. Among the novel enkephalins Ile-enkephalin (Tyr-Gly-Gly-Phe-Ile) was primarily observed in the African clawed frog (Xenopus laevis) PDYNs, while the structure of Phe-enkephalin (Tyr-Gly-Gly-Phe-Phe) was predicted by analyzing brain cDNA sequences encoding a PDYN of the African lungfish (Protopterus annectens). Ile-enkephalin can also be found in the PDYNs of four other fish species including the eel, bichir, zebrafish and tilapia, but no further occurrence for the Pheenkephalin motif is available as yet. Based on sequencing data, the biological relevance of Phe- and Ile-enkephalin is suggested, because both of them can arise by regular post-translational enzymatic processing of the respective neuropeptide precursors. In various receptor binding assays performed on rat brain membrane preparations both of the new peptides turned out to be moderate affinity opioids with a weak preference for the delta-opioid receptor (DOP) sites. Pheenkephalin of the lungfish displayed rather unexpectedly low affinities toward the mu-oploid receptor (MOP) and DOP, while exhibiting moderate affinity toward the K-opioid receptor (KOP). In receptor-mediated G-protein activation assays measured by the stimulation of [S-35]GTP gamma S binding, Met-enkephalin produced the highest stimulation followed by Leuenkephalin, Ile-enkephalin and Phe-enkephalin, whereas the least efficacious among these endogenous peptides was still more effective than the prototype opiate agonist morphine in these functional tests. (c) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.