Isolation and preliminary biological assessment of AADGAPLIRFamide and SVPGVLRFamide from Caenorhabditis elegans

To date, 9 FMRFamide-related peptides (FaRPs) have been structurally charac terised from Caenorhabditis elegans. Radioimmunometrical screening of an et hanolic extract of C. elegans revealed the presence of two additional FaRPs that were purified by reverse-phase HPLC and subjected to Edman degradatio n analysis and gas-phase sequencing. Unequivocal primary structures for the two FaRPs were determined as Ala-Ala-Asp-Gly-Ala-Pro-Leu-Ile-Arg-Phe-NH2 a nd Ser-Val-Pro-Gly-Val-Leu-Arg-Phe-NH2. Using MALDI-TOF mass. spectrometry, the molecular masses of the peptides were found to be 1032 Da (MH) and 875 Da (MH)(+), respectively. Two copies of AADGAPLIRFamide are predicted to b e encoded on the precursor gene termed flp-13, while one copy of SVPGVLRFam ide is located on flp-18. Synthetic replicates of the peptides were tested on Ascaris suum somatic muscle to assess bioactivity. ADDGAPLIRFamide had i nhibitory effects on A. suum muscle strips, which occurred over a range of concentrations from a threshold for activity of 10 nM to 10 muM. SVPGVLRFam ide was excitatory on A. suum somatic musculature from a threshold concentr ation for activity of 1 nM to 10 muM. The inhibitory and excitatory effects of AADGAPLIRFamide and SVPGVLRFamide, respectively, were the same for dors al and ventral muscle strips as well as innervated and denervated preparati ons, suggesting that these physiological effects are not nerve cord depende nt. Addition of ADDGAPLIRFamide (10 muM) to muscle strips preincubated in h igh-K+ and -Ca2+-free medium resulted in a normal inhibitory response. Pept ide addition to muscle strips preincubated in Cl--free medium showed no inh ibitory response, suggesting that the inhibitory response of the peptide ma y be chloride mediated. A normal excitatory response was noted following th e addition of 10 muM SVPGVLRFamide to muscle strips preincubated in high-K, Ca2+- and Cl--free media. (C) 2001 Academic Press.