Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays.
Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.
Chougule
Nanasaheb P
NP
School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UK. n.p.chougule@durham.ac.uk
Doyle
Emily
E
Fitches
Elaine
E
Gatehouse
John A
JA
D000818:Animals; D041981:Gastrointestinal Tract / Q000201:enzymology; D007814:Larva / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development; D009036:Moths / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D012697:Serine Endopeptidases / Q000378:metabolism*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology*
D014360:Trypsin Inhibitor, Kunitz Soybean; D012697:Serine Endopeptidases