Abstract
We report the isolation, characterization, and total synthesis of a small peptide ligand for nicotinic acetylcholine receptors (nAChRs). It is highly active against the neuromuscular receptor in frog but not in mice. In contrast, it induces seizures when injected centrally in mice and rats, suggesting that it may target neuronal nAChRs in mammals. Although such receptors may be important in both normal cognition and the pathophysiology of several neuropsychiatric disorders, there are few ligands to discriminate between the multiple receptor subtypes. The new peptide is a highly divergent alpha-conotoxin from the snail Conus imperialis, which preys on polychaete worms. In this article, the purification, structural analysis, synthesis, and preliminary physiological characterization of alpha-conotoxin ImI (alpha-CTx-ImI) are reported. The sequence of the peptide is: Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2. The peptide shows striking sequence differences from all alpha-conotoxins of fish-hunting Conus, but its disulfide-bridging is similar: [2-8; 3-12]. We suggest that cone venoms may provide an array of ligands with selectivity for various neuronal nAChR subtypes.
Highlights
From the Departments of $Psychiatry and $Biology, Uniuersity of Utah, Salt Luke City, Utah 84112 and the IIClayton
Conus is a largegenus of predatory snails that feed on fish, Thenewpeptide is ahighlydivergenta-conotoxin snails, and marine worms
We describe the purification and identity with natural peptide wavserified by co-elution experiments on characterization of the first a-conotoxin isolated from a ver- HPLC and capillary zone electrophoresis (CZE),by mass spectrometry, mivorous cone snail, Conus imperialis,and demonstrate that it and by disulfide “fingerprinting”(below)
Summary
After 2-5 min the reaction mixture was diluted with0.1% trifluoroacetic acid to decrease acetonitrile concentration and immediately injected. Monocyclic intermediates were alkylated with 100mg of iodoaccaptoethanolwas from PierceChemical Co.; dithiothreitolwas from etamide using the rapid alkylation method[24]. Labeled peptides were Boehringer Mannheim; trifluoroacetiaccid (sequencing grade) wafsrom submitted to sequence analysis to determine the locations of S-carbox-. Aldrich; acetonitrile ( U V grade) was from Baxter (Muskegon, MI). Tris(2-carboxyethyl)phosphine(TCEP) was synthesized by the method of Burns et al [20]. Disulfide Fingerprinting-Approximately 200 pmol of peptide in 50 pl of HPLC eluent werme ixed with 50 pl TCEP (20mM in 0.2M citrate, pH 3). After brief incubation at room temperature, the mixtures were analyzed by HPLC, to compare product distributions of natural and synthetic materials
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