Oleamide is an endogenous sleep-inducing lipid that has been isolated from the cerebrospinal fluid of sleep-deprived mammals. Oleamide is the best-understood member of the primary fatty acid amide family. One key unanswered question regarding oleamide and all other primary acid amides is the pathway by which these molecules are produced. One proposed pathway involves oleoyl-CoA and N-oleoylglycine as intermediates: oleic acid --> oleoyl-CoA --> N-oleoylglycine --> oleamide. The first and third reactions are known reactions, catalyzed by acyl-CoA synthetase and peptidylglycine alpha-amidating monooxygenase (PAM). Oleoyl-CoA formation from oleic acid has been demonstrated in vitro and in vivo while, to date, N-oleoylglycine cleavage to oleamide has been established only in vitro. PAM catalyzes the final step in alpha-amidated peptide biosynthesis, and its proposed role in primary fatty acid amide biosynthesis has been controversial. Mouse neuroblastoma N(18)TG(2) cells are an excellent model system for the study of oleamide biosynthesis because these cells convert [(14)C]-oleic acid to [(14)C]-oleamide and express PAM in a regulated fashion. We report herein that growth of the N(18)TG(2) cells in the presence of [(14)C]-oleic acid under conditions known to stimulate PAM expression generates an increase in [(14)C]-oleamide or in the presence of a PAM inhibitor generates [(14)C]-N-oleoylglycine. This represents the first identification of N-oleoylglycine from a biological source. In addition, N(18)TG(2) cell growth in the presence of N-oleoylglycine yields oleamide. These results strongly indicate that N-oleoylglycine is an intermediate in oleamide biosynthesis and provide further evidence that PAM does have a role in primary fatty acid amide production in vivo.

Cone snails comprise approximately 500 species of venomous molluscs which have evolved the ability to generate multiple toxins with varied and exquisite selectivity. α-Conotoxin is a powerful tool for defining the composition and function of nicotinic acetylcholine receptors which play a crucial role in excitatory neurotransmission and are important targets for drugs and insecticides. An α4/7 conotoxin, Lp1.1, originally identified by cDNA and genomic DNA cloning from Conus leopardus, was found devoid of the highly conserved Pro residue in the first intercysteine loop. To further study this toxin, α-Lp1.1 was chemically synthesized and refolded into its globular disulfide isomer. The synthetic Lp1.1 induced seizure and paralysis on freshwater goldfish and selectively reversibly inhibited ACh-evoked currents in Xenopus oocytes expressing rat α3β2 and α6α3β2 nAChRs. Comparing the distinct primary structure with other functionally related α-conotoxins could indicate structural features in Lp1.1 that may be associated with its unique receptor recognition profile.

Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. alpha3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of alpha3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac1.1a and Ac1.1b, with appropriate disulfide bonding, based on cDNA sequences of alpha3/5 conotoxins from C. achatinus. Ac1.1a and Ac1.1b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC50 values of 36 nM and 26 nM, respectively. For Ac1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the N-terminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the alpha1-delta subunit interface instead of the alpha1-gamma binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor.