Worm Breeder's Gazette 10(3): 155
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
AF1 was isolated from an acid methanol extract of 10,000 Ascaris heads. The purification procedure consisted of a two-step fractionation with C18 cartridges, reverse phase HPLC (RP-HPLC) using three solvent systems, and HPLC gel filtration. Separations were monitored with a FMRFamide radioimmunoassay (RIA), using an antiserum provided by R. Calabrese. Material eluted from C18 cartridges with 50% acetonitrile in aqueous trifluoroacetic acid (TFA) was separated into several immunoreactive peaks by gradient RP-HPLC with butanol in aqueous TFA. Further purification was carried out by gradient RP-HPLC with acetonitrile in aqueous TFA. Subsequent HPLC gel filtration yielded several peaks of optical density not corresponding to the peak of immunoreactivity; in separate runs the active peak eluted earlier than FMRFamide, suggesting that the Ascaris peptide is bigger. The final step was isocratic RP-HPLC with acetonitrile in aqueous heptafluorobutyric acid (HFBA) yielding several peaks of optical density, one of which corresponded to the active peak. Half (55 pmoles) of the purified peptide was taken for amino acid sequence determination on a liquid phase sequenator, and gave the sequence KNEFIRF. Amidation of the C-terminus was indicated by the specificity of the antiserum used for RIA. Synthetic KNEFIRFamide ( sequence determination and peptide synthesis were performed by the Biotechnology Center, University of Wisconsin) coeluted with the natural peptide by HPLC gel filtration and by isocratic RP-HPLC. Since FMRFamide was first described, more than 20 FMRFamide-like neuropeptides have been isolated or predicted from a gene sequence. Only one of these, MDSNFIRFamide, predicted from the Drosophila FMRFamide gene, also has a C-terminal IRFamide. Four additional FMRFamide-like neuropeptides have been isolated from Ascaris, yielding two complete sequences and two partial sequences; these four peptides are also structurally different from previously known FMRFamidelike neuropeptides. Hence, it is clear that there exists a family of FMRFamide-like neuropeptides in Ascaris and it will now be possible to assess their physiological roles. In the cases from other phyla that have been analyzed, different isoforms of FMRFamide had quantitatively or qualitatively different physiological activities. There may also be diversity of action of the Ascaris FMRFamidelike neuropeptides. One action of AF1 seems to be inhibition of locomotory movements. Injection of 0.1 ml 10+E-6 M AF1 into the anterior portion of Ascaris ( N=15) blocks locomotory movements in the region of the injection. The physiological effects of AF1 on the Ascaris motornervous system were explored using intracellular recording techniques. AF1 exerts a dramatic effect on the electrical properties of the ventral inhibitory (VI) motorneurons: slow oscillatory potentials, whether spontaneous or induced by depolarization, were rapidly and reversibly blocked. Consistent block was obtained at a concentration of 10+E-7 M (5 of 5 preparations) with some preparations sensitive at a concentration as low as 10+E-9 M (5 of 12). AF1 will be useful in analyzing the physiological mechanisms underlying membrane potential oscillations in Ascaris.