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‐α‐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. Copyright © 1994, Wiley Blackwell. All rights reserved