Endopeptidase activity of larval Lacanobia oleracea corpus allatum: metabolism of Manduca sexta allatostatin and allatotropin.
The degradation of synthetic Manduca sexta allatostatin (Manse-AS) and allatotropin (Manse-AT) by enzymes associated with the corpus allatum (CA) of larvae of the tomato moth, Lacanobia oleracea, was investigated using reversed-phase high performance liquid chromatography and matrix-assisted laser desorption ionisation-time of flight mass spectrometry. Manduca sexta allatostatin was metabolised by CA extract to Manse-AS5-15, Manse-AS6-15, and Manse-AS7-15, which indicates enzymic cleavage at the C-terminal side of arginine residues R3 and R5 and the N-terminal side of R5, suggesting this is due to a trypsin-like enzyme. In support of this, the same degradation products were identified after Manse-AS was incubated with trypsin, and CA enzymic activity could be inhibited up to 79% by aprotinin. Degradation of Manse-AT by CA extract was also trypsin-like, cleaving at the C-terminal side of the basic residues K3 and R11 to produce Manse-AT4-13 and Manse-AT1-11. Metabolism by trypsin produced the same deletion peptides, but the major product due to this enzyme was Manse-AT4-11. Hydrolysis of Manse-AT by CA could only be partially inhibited by high doses of aprotinin (36%), and the CA extract also cleaved Manse-AT between M8 and T9 to produce Manse-AT1-8. A trypsin-like peptidase appears to be the major enzyme present in the CA of larval L. oleracea that acts to metabolise Manse-AS and Manse-AT. In addition, an unidentified enzyme that cleaves between M and T residues degraded Manse-AT.
Audsley
Neil
N
Central Science Laboratory, Sand Hutton, York, United Kingdom. n.audsley@csl.gov.uk
Weaver
Robert J
RJ
D000818:Animals; D007611:Aprotinin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003335:Corpora Allata / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism*; D007301:Insect Hormones / Q000378:metabolism*; D019476:Insect Proteins / Q000378:metabolism*; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D018402:Manduca / Q000737:chemistry; D013058:Mass Spectrometry; D009036:Moths / Q000201:enzymology*; D009479:Neuropeptides / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*
D007301:Insect Hormones; D019476:Insect Proteins; D009479:Neuropeptides; D010455:Peptides; C071432:allatostatin protein, Manduca sexta; C050669:allatotropin; D007611:Aprotinin; D010450:Endopeptidases; D011761:Pyrrolidonecarboxylic Acid