In Vitro Biosynthesis of Locust Adipokinetic Hormones: Isolation and Identification of the Bioactive Peptides and Their Prohormones
The biosynthesis of adipokinetic hormones (AKH) I and II and their precursors was studied in the migratory locust Locusta migratoria using radiolabeled amino acids in an in vitro system. The peptides from the glandular part of the corpora cardiaca (CC) were extracted and separated by reversed-phase high performance liquid chromatography (HPLC). Incorporation of the [3H] amino acids was measured by monitoring the radioactivity of the column eluate. Total time for biosynthesis and processing of the prohormones to the bioactive AKH I and II was 75 min. Only the glandular part of the CC synthesized these peptide hormones.
Protein fractions from CC were separated by gradient gel electrophoresis (PAGE or SDS-PAGE) and by two-dimensional gel electrophoresis (isoelectrofocusing followed by SDS-PAGE). After electrophoresis, proteins were transferred to nitrocellulose membranes by electroblotting and the incorporation of radiolabel was measured with an automatic linear analyzer. Time-course studies and pulse-labeling experiments revealed incorporation of radiolabel into most proteins.
By using monoclonal antibodies raised against AKH I, immunoreactive protein bands (prohormones and/or processing products) with a Mr of 72,000 and 33,000 were only detectable in the cytoplasm fraction of the glandular lobe of the CC.
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