Trypsin-like activity of membrane-bound midgut proteases from Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Membrane-bound proteases from preparations of the midgut of 5 super(th) instar velvetbean caterpillars, Anticarsia gemmatalis (Huebner) 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- alpha -benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and N- alpha -p-tosyl-L-Arg methyl ester (L-TAME). Higher activities were observed at 50 degree 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 chloromethyl ketone (TLCK), benzamidine and aprotinin. TLCK and benzamidine were particularly active inhibitors. The K sub(M)-values obtained were 0.23 mM for L-BApNA and 92.5 mu M 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.