Abstract
Keywords. Trypsin, proteases, velvetbean caterpillar, Lepidoptera, Noctuidae, Anticarsia Abstract. Membrane-bound proteases from preparations of the midgut of 5 th instar velvetbean caterpillars, Anticarsia gemmatalis (Hubner) were obtained by resuspension of the pellet obtained after 100,000 g centrifugation. As expected of trypsin-like proteases, they hydrolyzed casein and the synthetic substrates N-D -benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and N-D -p-tosyl-L-Arg methyl ester (L-TAME). Higher activities were observed at 50°C, and at pH 8.5 and 8.0 for both synthetic substrates L-BApNA and L-TAME. The membrane-bound proteases were inhibited by EDTA, phenylmethan sulphonyl fluoride (PMSF), tosyl-L-lysine chlo- romethyl ketone (TLCK), benzamidine and aprotinin. TLCK and benzamidine were particularly active inhibitors. The KM-values obtained were 0.23 mM for L-BApNA and 92.5 µM for L-TAME. These results provide evidence for the presence of membrane- bound trypsin-like proteases in the midgut of the velvetbean caterpillar, a key soybean pest in warm climates. The interaction between A. gemmatalis digestive proteases and soybean protease inhibitors has potentially important consequences for soybean breeding programs.
Highlights
An understanding of insect-plant interactions is important for predicting host plant colonization by insect-pests and for developing alternative control methods (Pedigo, 1989; Boulter, 1993; Bernays & Chapman, 1994; Carlini & Grossi-de-Sá, 2002)
The first study of the mechanism of enzyme secretion in Lepidoptera was done using larvae of Bombyx mori and indicated that membrane-bound trypsin-like proteases are transported from the tissues to the lumen of the gut where they are solubilized and converted into an active form
Some of the trypsin-like proteases are incorporated in the peritrophic membrane, an extracellular sheath which protects the midgut epithelial cells (Eguchi et al, 1982; Kuriyama & Eguchi, 1985)
Summary
An understanding of insect-plant interactions is important for predicting host plant colonization by insect-pests and for developing alternative control methods (Pedigo, 1989; Boulter, 1993; Bernays & Chapman, 1994; Carlini & Grossi-de-Sá, 2002). Plants usually contain an array of proteins toxic to insects, the legumes that are rich in nitrogen (Bernays & Chapman, 1994; Koiwa et al, 1997; Hilder & Boulter, 1999; Stotz et al, 1999; Carlini & Grossi-de-Sá, 2002). Serine proteases are the best-studied proteases and are present in viruses, prokaryotes and eukaryotes, suggesting they are vital for the survival of organisms. They are frequently recorded in insects, Lepidoptera, where they are the main family of proteases (Terra & Ferreira, 1994). The most frequently studied serine proteases, trypsin- and chymotrypsin-like, act in a wide range of physiological processes including digestion, protein activation in the melanization cascade, antibacterial activity and insect immune response (Nakajima et al, 1997; Gorman et al, 2000a,b; Ma & Kanost, 2000). Jordão et al (1999) suggest that the trypsin from Spodoptera frugiperda (Lepidoptera: Noctuidae) is probably processed
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