Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization, cDNA cloning, and expressed sequence tag analysis.
The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.
Hegedus
Dwayne
D
Agriculture and Agri-Food Canada, Saskatoon, Canada. hegedusd@em.agr.ca
Baldwin
Doug
D
O'Grady
Michael
M
Braun
Lorraine
L
Gleddie
Steve
S
Sharpe
Andrew
A
Lydiate
Derek
D
Erlandson
Martin
M
D000595:Amino Acid Sequence; D000824:Animal Nutritional Physiological Phenomena; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004064:Digestive System / Q000033:anatomy & histology / Q000187:drug effects / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000494:pharmacology
D018076:DNA, Complementary; D011480:Protease Inhibitors; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D012697:Serine Endopeptidases