title: Degradation of Manduca sexta allatostatin and allatotropin by proteases associated with the foregut of Lacanobia oleracea larvae.
abstract: The degradation of synthetic Manduca sexta allatostatin (Manse-AS) and allatotropin (Manse-AT), by enzymes of the foregut of larvae of the tomato moth, Lacanobia oleracea was investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Edman sequencing. Metabolism of 1nmol Manse-AS by foregut extract (1microg protein) was rapid, t(1/2) approximately 5min, with two major products produced. Mass spectrometry of HPLC fractions identified cleavage products Manse-AS-(4-15) and Manse-AS-(6-15), which indicates enzymatic cleavage at the C-terminal side of arginine residues (R(3) and R(5)). This degradation of Manse-AS could be inhibited by up to 80% by the serine protease inhibitor aprotinin, but not PMSF, pepstatin, E64, EDTA, or 1,10-phenanthroline.M. sexta allatotropin was also rapidly degraded when incubated with foregut extract, t(1/2) approximately 8min, producing two metabolic products, one of which was identified as Manse-AT-(1-11), showing enzymatic cleavage at the C-terminal side of arginine (R(11)). The second product was identified as Manse-AT-(1-8). Hydrolysis of Manse-AT could only be partially inhibited by high doses of aprotinin (30%).
authors.affiliation: Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK. n.audsley@csl.gov.uk
authors.forename: Neil
authors.initials: N
authors.lastname: Audsley
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Weaver
authors.forename: John P
authors.initials: JP
authors.lastname: Edwards
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004064:Digestive System / Q000201:enzymology*; D006868:Hydrolysis; D007301:Insect Hormones / Q000378:metabolism*; D007814:Larva / Q000201:enzymology*; D018402:Manduca; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology* / Q000254:growth & development; D009479:Neuropeptides / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D007301:Insect Hormones; D009479:Neuropeptides; C076523:allatostatin; C050669:allatotropin