Isolation and characterization of two digestive trypsin-like proteinases from larvae of the stalk corn borer, Sesamia nonagrioides.
Two digestive trypsin-like proteinases from Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae) larvae were purified by benzamidine-Sepharose affinity chromatography. The purified enzymes showed molecular size of 27 (trypsin-I) and 24 KDa (trypsin-II). Amino acid analysis and N-terminal sequencing confirmed their relationship with other trypsins from lepidopteran larvae. However, trypsin-I presented one lysine at position 11, being the first report of this amino acid in the sequence of a lepidopteran digestive trypsin. Trypsin-I had an isoelectric point of 6.0, and a Km of 2.2 x 10(-4) M and 3.9 x 10(-5) M for BApNa and BAEE, respectively. Trypsin-II presented an isoelectric point of 8.7, and Km values of 1.7 x 10(-4) M (BApNa) and 3.8 x 10(-5) M (BAEE). Both enzymes were differentially inhibited by some proteinase inhibitors. In particular, trypsin-I was inhibited by E-64 (ID50 = 6 microM) but not by lima bean trypsin inhibitor (LBI), whereas trypsin-II was inhibited by LBI (ID50 = 1 microM) and poorly by E-64 (ID50 = 85 microM). Changes in the susceptibility of the trypsin-like activity of midgut extracts from different larval instars to these inhibitors suggest that the relative proportion of these two enzymes varied through larval development, being predominant in early instars trypsin-I and in late instars trypsin-II.
Novillo
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C
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Castañera
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P
Ortego
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F
D000818:Animals; D004064:Digestive System / Q000201:enzymology; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007814:Larva / Q000201:enzymology; D007915:Lepidoptera / Q000201:enzymology*; D010940:Plant Proteins / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism
D010940:Plant Proteins; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; D010450:Endopeptidases; D014357:Trypsin