Leonardo L.P. Macedo

Identification of a Kunitz-type proteinase inhibitor from Pithecellobium dumosum seeds with insecticidal properties and double activity.

A trypsin inhibitor, PdKI, was purified from Pithecellobium dumosum seeds by TCA precipitation, trypsin-sepharose chromatography, and reversed-phase-HPLC. PdKI was purified 217.6-fold and recovered 4.7%. SDS-PAGE showed that PdKI is a single polypeptide chain of 18.9 kDa and 19.7 kDa by MALDI-TOF. The inhibition on trypsin was stable in the pH range 2-10 and at a temperature of 50 degrees C. The Ki values were 3.56 x 10(-8)and 7.61 x 10(-7) M with competitive and noncompetitive inhibition mechanisms for trypsin and papain, respectively. The N-terminal sequence identified with members of Kunitz-type inhibitors from the Mimosoideae and Caesalpinoideae subfamilies. PdKI was effective against digestive proteinase from Zabrotes subfasciatus, Ceratitis capitata, Plodia interpunctella, Alabama argillaceae, and Callosobruchus maculatus, with 69, 66, 44, 38, and 29% inhibition, respectively. Results support that PdKI is a member of the Kunitz inhibitor family and its insecticidal properties indicate a potent insect antifeedant.

Proteolytic Digestive Enzymes and Peritrophic Membranes during the Development of Plodia interpunctella (Lepidoptera: Piralidae): Targets for the action of Soybean Trypsin Inhibitor (SBTI) and Chitin-Binding Vicilin (EvV)

The digestive system of P. interpunctella was characterized during its larval development to determine possible targets for the action of proteinaceous enzyme inhibitors and chitin-binding proteins. High proteolytic activities using azocasein at pH 9.5 as substrate were found. These specific enzymatic activities (AU/mg protein) showed an increase in the homogenate of third instar larvae, and when analyzed by individual larvae (AU/gut), the increase was in sixth instar larvae. Zymograms showed two bands corresponding to those enzymatic activities, which were inhibited by TLCK and SBTI, indicating that the larvae mainly used serine proteinases at pH 9.5 in their digestive process. The presence of a peritrophic membrane in the larvae was confirmed by chemical testing and light microscopy. In a bioassay, P. interpunctella was not susceptible to the soybean trypsin inhibitor, which did not affect larval mass and mortality, likely due to the weak association with its target digestive enzyme. EvV (Erythrina velutina vicilin), when added to the diet, affected mortality (LD50 0.23%) and larval mass (ED50 0.27%). This effect was associated with EvV-binding to the peritrophic membrane, as seen by immunolocalization. EvV was susceptible to gut enzymes and after the digestion process, released an immunoreactive fragment that was bound to the peritrophic matrix, which probably was responsible for the action of EvV.

Larvicidal Effects of a Chitin-Binding Vicilin from Erythrina velutina Seeds on the Mediterranean Fruit Fly Ceratitis capitata

Chitin-binding vicilin from Erythrina velutina seeds was purified by ammonium sulfate followed by affinity chromatography on a chitin column and gel filtration on Superose-6-10-300-GL. The Erythrina velutina vicilin, called EvV, is a tetrameric glycoprotein composed of 1.85% carbohydrates and M r of 216.6 kDa, consisting of two subunits of M r of 54.8 and two subunits of M r of 50.8 kDa. The EvV homogeneity was confirmed in native PAGE where it was observed to be a unique acid-protein band with slow mobility in this gel. Effect of EvV on C. capitata larvae was examined by bioassay and its mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV when added to diet caused strong effect on mortality (ED50 of 0.14%) and larval mass (WD50 of 0.12%). These deleterious effects were associated to the binding to chitin structures present in peritrophic membrane and to gut epithelial cells, and its low digestibility in C. capitata digestive tract. These results are the first demonstration of a proteinaceous bioinsecticide from plant origin effective against C. capitata larvae. EvV may be part of the pest management programs or an alternative in plant improvement program.

Effects of a chitin binding vicilin from Erythrina velutina seeds on bean bruchid pests (Callosobruchus maculatus and Zabrotes subfasciatus).

Erythrina velutina vicilin, EvV, is a dimeric glycoprotein with Mr of 124.6 kDa. EvV was tested for anti-insect activity against bean bruchid larvae. EvV had LD(50) of 0.10% and ED(50) of 0.14% for Z. subfasciatus and LD(50) of 0.26% and ED(50) of 0.19% for C. maculatus. EvV was not digested by bean larvae enzymes until 12 h of incubation, and at 24 h EvV was more resistant to Z. subfasciatus enzymes.