N-acylation of Aplysia egg-laying hormone with biotin: Characterization of bioactive and inactive derivatives

Susan L. Knock, Brian T. Miller, James E. Blankenship, Gregg T. Nagle, John S. Smith, Alexander Kurosky

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    Chemical modification of the egg-laying hormone (ELH) of Aplysia by reaction with the N-hydroxysuccinimide ester of biotin, which contained 6-aminohexanoic acid as spacer, yielded seven distinct derivatives that were readily separated by reversed-phase high performance liquid chromatography. The derivatives were chemically characterized by amino acid compositional analysis, sequence analysis, and mass spectrometry. The seven derivatives resulted from combinations of differential modification of the three amino groups in the ELH molecule located at Ile1 (α-NH2), Lys8 and Lys36. Of the seven derivatives formed, only one, monobiotinyl Lys36-ELH, was biologically active in eliciting egg-laying activity and altering the electrophysiological activity of the abdominal ganglion neuron R15 and LB and LC cluster neurons. In addition, evaluation of the time course of biotinylation of ELH revealed that the relative rate of amino group reactivity was ∈-NH2-Lys36 > ∈-NH2-Lys8 ≫ α-NH2-Ile1. The slow rate of reaction of the terminal α-amino group suggested that it was relatively inaccessible to biotinylation, possibly due to conformational factors or to ion-pair formation with an unidentified carboxyl group. Loss of bioactivity of ELH monobiotinylated on the α-amino group, coupled with the unusually low reactivity of the α-amino group, provided strong evidence for the importance of the α-amino group in ELH function. Furthermore, the development and availability of a bioactive ELH probe should greatly facilitate the isolation, characterization, and localization of the ELH receptor.

    Original languageEnglish (US)
    Pages (from-to)24413-24419
    Number of pages7
    JournalJournal of Biological Chemistry
    Issue number36
    StatePublished - Dec 25 1991


    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Knock, S. L., Miller, B. T., Blankenship, J. E., Nagle, G. T., Smith, J. S., & Kurosky, A. (1991). N-acylation of Aplysia egg-laying hormone with biotin: Characterization of bioactive and inactive derivatives. Journal of Biological Chemistry, 266(36), 24413-24419.