Isolation and Preliminary Biological Assessment of AADGAPLIRFamide and SVPGVLRFamide from Caenorhabditis elegans

Abstract

To date, 9 FMRFamide-related peptides (FaRPs) have been structurally characterised from Caenorhabditis elegans. Radioimmunometrical screening of an ethanolic extract of C. elegans revealed the presence of two additional FaRPs that were purified by reverse-phase HPLC and subjected to Edman degradation 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 and 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 be encoded on the precursor gene termed flp-13, while one copy of SVPGVLRFamide is located on flp-18. Synthetic replicates of the peptides were tested on Ascaris suum somatic muscle to assess bioactivity. ADDGAPLIRFamide had inhibitory effects on A. suum muscle strips, which occurred over a range of concentrations from a threshold for activity of 10 nM to 10 μM. SVPGVLRFamide was excitatory on A. suum somatic musculature from a threshold concentration for activity of 1 nM to 10 μM. The inhibitory and excitatory effects of AADGAPLIRFamide and SVPGVLRFamide, respectively, were the same for dorsal and ventral muscle strips as well as innervated and denervated preparations, suggesting that these physiological effects are not nerve cord dependent. Addition of ADDGAPLIRFamide (10 μM) to muscle strips preincubated in high-K⁺ and -Ca²⁺-free medium resulted in a normal inhibitory response. Peptide addition to muscle strips preincubated in Cl⁻-free medium showed no inhibitory response, suggesting that the inhibitory response of the peptide may be chloride mediated. A normal excitatory response was noted following the addition of 10 μM SVPGVLRFamide to muscle strips preincubated in high-K⁺, Ca²⁺- and Cl⁻-free media.