Purification of a functional competitive antagonist for calcitonin gene related peptide action from sardine hydrolysates

Abstract

Calcitonin gene related peptide (CGRP) related molecules were purified from sardine hydrolysates prepared using 0.1% alcalase and two hours of hydrolysis. Gel exclusion chromatography and HPLC performed purification of these molecules. The purified molecules were characterised using specific CGRP radioimmunoassays and radioreceptoraasays. From 22 mg of crude extract, we obtained 14 µg of CGRP related molecules, the molecular weight determined by mass spectrophotometry was 6000 daltons. The biological activity of these molecules was analysed using the ability of CGRP to stimulate the adenylate cyclase activity in rat liver membranes. The purified molecules induced an inhibition of the CGRP stimulated adenylate cyclase activity, this effect was specific as no such effect was observed on the glucagon stimulated adenylate cyclase activity measured in the same rat liver membrane preparation. These results suggest that the purified molecules may act as antagonists for peptides that bind to CGRP receptors in rat liver membranes. These new antagonists may be of particular importance in various aspects of CGRP action in vertebrates.