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. (Lepi doptera: Noctuidae) larvae were purified by benzamidine-Sepharose affinity chromatography. The purified enzymes showed molecular sizes of 27 (trypsin- I) and 24 KDa (trypsin-II). Amino acid analysis and N-terminal sequencing c onfirmed their relationship with other trypsins from lepidopteran larvae. H owever, trypsin-I presented one lysine at position 11, being the first repo rt 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 2.2x10(-4) M and 3.9 x10(-5) M for BApNa and BAEE, respectively. Trypsin-II presented an isoelec tric point of 8.7, and Km values of 1.7x10(-4) M (BApNa) and 3.8x10(-5) M ( BAEE). Both enzymes were differentially inhibited by some proteinase inhibi tors. In particular, trypsin-I was inhibited by E-64 (ID50=6 mu M) but not by lima bean trypsin inhibitor (LBI), whereas trypsin-IT was inhibited by L BI (ID50=1 mu M) and poorly by E-64 (ID50=85 mu M). Changes in the suscepti bility of the trypsin-like activity of midgut extracts from different larva l 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. (C) 1999 Elsevier Science Ltd. All rights reserved.