Novel FMRFamide-like neuropeptides from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii

Abstract

Isolation of the FMRFamide-like neuropeptides (FLPs) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii was performed using 5000 ground eyestalks extracted in methanol/acetic acid/water (90:1:9). After the extract was partially purified using C18 cartridges, it was further purified by eight steps of RP-HPLC using four kinds of columns: C18, C8, cyano and phenyl, and three solvent systems: acetonitrile (ACN)/trifluoroacetic acid, ACN/heptafluoroacetic acid and ACN/triethylammonium acetate. Dot-ELISA, using a mixture of two monoclonal antibodies, FM-23 (made against FMRFamide) and AF1-62 (made against KNEFIRFamide), was used to monitor FLPs in the fractions during the purification processes. Two subfamilies of five FLPs were obtained from the first six fractions of the first step of purification by RP-HPLC; four of them are new sequences. Three FLPs, DRNFLRFamide, ADKNFLRFamide and NYDKNFLRFamide share five to seven common C-terminus residues with those found in the pericardial organs of Homarus americanus, Callinectes sapidus and Procambarus clarkii. The other two FLPs, APALRLRFamide and DRTPALRLRFamide, share six common residues at the C-terminus with those found in Limulus polyphemus and Periplaneta americana. These findings suggest that more than one transcript might be responsible for encoding FLPs in M. rosenbergii. Copyright (C) 1998 Elsevier Science Inc.