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, A Sequences Bioactive Neuropeptide Isolated from the Nematode, Ascaris suum

Cynthia Cowden, Antony O.W. Stretton and Ralph E. Davis

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