title: Reconstitution of adipokinetic hormone biosynthesis in vitro indicates steps in prohormone processing.
abstract: We have used a complete, synthetic precursor to adipokinetic hormone I (AKH I) and oligopeptides derived from this precursor as substrates for prohormone-processing enzymes extracted from AKH-synthesizing neurosecretory cells to reconstitute the post-translational steps in AKH biosynthesis in vitro. The results demonstrate the existence of endoproteolytic activity which cleaves the precursor only at the appropriate processing site (at the C-terminal side of Arg13). Further proteolytic processing of C-terminally extended AKH I (AKH-Gly-Lys-Arg) by a carboxypeptidase H-like activity removes the basic residues producing AKH-Gly-Lys, followed by AKH-Gly. Finally, a peptidylglycine-alpha-amidating-monooxygenase activity produces the amidated bioactive product from the glycine-extended peptide in a two-step process, the first of which requires ascorbate and Cu2+. Our results show that all steps in AKH precursor processing can be reconstituted and studied in vitro, providing a system to characterize the processing enzymes and to investigate the development of enzyme inhibitors for use as potential insecticides.
authors.affiliation: Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, England.
authors.forename: R C
authors.initials: RC
authors.lastname: Rayne
authors.forename: M
authors.initials: M
authors.lastname: O'Shea
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D006110:Grasshoppers; D066298:In Vitro Techniques; D007301:Insect Hormones / Q000096:biosynthesis* / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009490:Neurosecretory Systems / Q000201:enzymology*; D009842:Oligopeptides / Q000096:biosynthesis* / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D013379:Substrate Specificity
chemical_list: D007301:Insect Hormones; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011480:Protease Inhibitors; D011498:Protein Precursors; C044833:adipokinetic hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D002268:Carboxypeptidases; D010450:Endopeptidases; D011761:Pyrrolidonecarboxylic Acid