RECONSTITUTION OF ADIPOKINETIC HORMONE BIOSYNTHESIS IN-VITRO INDICATES STEPS IN PROHORMONE PROCESSING

We have used a complete, synthetic precursor to adipokinetic hormone I (AKH I) and oligopeptides derived from this precursor as substrates f or prohormone-processing enzymes extracted from AKH-synthesizing neuro secretory cells to reconstitute the post-translational steps in AKH bi osynthesis in vitro. The results demonstrate the existence of endoprot eolytic activity which cleaves the precursor only at the appropriate p rocessing site (at the C-terminal side of Arg13). Further proteolytic processing of C-terminally extended AKH I (AKH-Gly-Lys-Arg) by a carbo xypeptidase II-like activity removes the basic residues producing AKH- Gly-Lys, followed by AKH-Gly. Finally, a peptidylglycine-a: amidating- monooxygenase activity produces the amidated bioactive product from th e glycine-extended peptide in a two-step process, the first of which r equires ascorbate and Cu2+. Our results show that all steps in AKH pre cursor processing can be reconstituted and studied in vitro, providing a system to characterize the processing enzymes and to investigate th e development of enzyme inhibitors for use as potential insecticides.