ISOLATION OF AF2 (KHEYLRFAMIDE) FROM CAENORHABDITIS-ELEGANS - EVIDENCE FOR THE PRESENCE OF MORE THAN ONE FMRFAMIDE RELATED PEPTIDE-ENCODINGGENE

Numerous FMRF amide-related peptides (FaRPs) have been isolated and se quenced from extracts of free-living and parasitic nematodes. The most abundant FaRP identified in ethanolic/methanolic extracts of the para sitic forms, Ascaris suum and Haemonchus contortus and from the free-l iving nematode, Panagrellus redivivus, was KHEYLRF amide (AF2). Analys is of the nucleotide sequences of cloned FaRP-precursor genes from C. elegans and, more recently, Caenorhabditis vulgaris identified a serie s of related FaRPs which did not include AF2. An acid-ethanol extract of Caenorhabditis elegans was screened radioimmunometrically for the p resence of FaRPs using a C-terminally directed FaRP antiserum. Approxi mately 300 pmols of the most abundant immunoreactive peptide was purif ied to homogeneity and 30 pmols was subjected to Edman degradation ana lysis and gas-phase sequencing. The unequivocal primary structure of t he heptapeptide, Lys-His-Glu-Tyr-Leu-Arg-Phe-NH2 (AF2) was determined following a single gas-phase sequencing run. The molecular mass of the peptide was determined using a time-of-flight mass spectrometer and w as found to be 920 (MH(+))(-), which was consistent with the theoretic al mass of C-terminally amidated AF2. These results indicate that C. e legans possesses more than one FaRP gene. (C) 1995 Academic Press, Inc .