4). Furthermore, the unaltered enzymatic activity is a strong indication that there were no major alterations in the protein structure due to the chemical modifications. Despite their highly conserved structures and catalytic mechanisms, little is known about the physiological role of ureases in the source organisms, especially in plants (Carlini and Polacco, 2008). The widespread distribution of ureases in leguminous seeds as well as the accumulation pattern of the protein during seed maturation is suggestive

of an important physiological role (Carlini and Polacco, 2008). Canatoxin, first isolated as a highly toxic protein (Carlini and Guimaraes, 1981) and later identified as an isoform of JBU (Follmer et al., 2001), displays insecticidal activity against insects of different orders (Carlini et al., 1997; Staniscuaski and Carlini, 2012; Staniscuaski et al., 2005). The entomotoxic property of CNTX is Ivacaftor cost independent of its enzymatic activity and involves both the intact BIBF 1120 ic50 protein and peptides released by the insect’s digestive enzymes, with a 10 kDa peptide representing the most toxic fragment (Ferreira-DaSilva et al., 2000). The more abundant isoform of urease, JBU, was as lethal as CNTX in feeding trials either with the cotton stainer bug D. peruvianus

( Follmer et al., 2004), the kissing bug R. prolixus ( Staniscuaski et al., 2009), or the milkweed bug Oncopeltus fasciatus ( Defferrari et al., 2011). The insecticidal activity towards D. peruvianus was partially affected for both JBU-Lys Parvulin and JBU-Ac, as compared to the native protein. It is known that one essential step in ureases insecticidal activity is their hydrolysis by the insects’ digestive enzymes ( Carlini et al., 1997; Defferrari et al., 2011; Ferreira-DaSilva et al., 2000; Piovesan et al., 2008). The results obtained showed that the modification of acidic residues affected the toxic property by blocking the release of the entomotoxic peptide(s) from the urease molecule. Analysis of the localization of the

toxic peptide, Jaburetox, within JBU structure shows two aspartic acid residues flanking up- and down-stream the peptide sequence. It has been previously demonstrated that JBU is hydrolyzed by D. peruvianus digestive enzymes preferentially between the residues Ala-228 and Asp-229, at the N-terminal region of Jaburetox, and between Arg-322 and Asp-323, at the C-terminal region ( Piovesan et al., 2008). Even though one of these residues (Asp-323) may not be accessible, the modification of a single Asp residue flanking the entomotoxic peptide could impair its release. It is also important to note that, according to the results presented here, JBU-Ac seems not to be hydrolyzed at all by the insect digestive enzymes. This result is consistent with previous observations that the main class(es) of D. peruvianus digestive enzymes hydrolyze bonds at the N- or C-terminal sides of aspartic acid residues ( Piovesan et al., 2008).