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Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: Characterization, cDNA cloning, and expressed sequence tag analysis
Author(s) -
Hegedus Dwayne,
Baldwin Doug,
O'Grady Michael,
Braun Lorraine,
Gleddie Steve,
Sharpe Andrew,
Lydiate Derek,
Erlandson Martin
Publication year - 2003
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.10084
Subject(s) - midgut , biology , proteases , biochemistry , protease , serine protease , complementary dna , microbiology and biotechnology , trypsin , serine , elastase , peptide sequence , carboxypeptidase , serine proteinase inhibitors , chymotrypsin , enzyme , gene , larva , botany
Abstract 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. Arch. Insect Biochem. Physiol. 53:30–47, 2003. © 2003 Wiley‐Liss, Inc.