, 2000). A recombinant peptide equivalent to pepcanatox was developed from the JBUre-II corresponding sequence, and named Jaburetox (Jackbean urease toxin) ( Mulinari et al., 2007). This peptide was lethal to several insects, such as Dysdercus peruvianus, Spodoptera frugiperda, Blattella germanica, Rhodnius prolixus and Triatoma infestans, but it was innocuous when injected or ingested by mice and neonate rats ( Mulinari

et al., 2007; Tomazetto et al., 2007). For simplicity reasons, the term Jaburetox will be used here as synonymous of C. ensiformis urease DNA Synthesis inhibitor entomotoxic peptides, regardless of their origin (JBU or JBUre-II). It is worth mention that, within the entomotoxic peptide region, JBU and JBUre-II present 74 and 82% of sequence identity and similarity, respectively. The mode of action of Jaburetox, as well

as that of urease, is not fully understood. JBU and Jaburetox are capable of altering the serotonin-induced secretion of insects Malpighian tubules, indicating an effect on the osmotic balance of the insects ( Staniscuaski et al., 2009), both ex vivo selleck products and in vivo ( Carlini et al., 1997). JBU also can alter the secretion and contraction patterns of the anterior midgut in R. prolixus ( Staniscuaski et al., 2010). Chemical modification of amino acids residues can provide essential information about protein structure and functions. For JBU, this approach has been used to demonstrate the influence of histidine residues in the copper-induced oligomerization of JBU, and how this affected its ureolytic and insecticidal activities (Follmer and Carlini, 2005). In this work, we have performed chemical modification of lysine, aspartic and glutamic acid residues of JBU aiming to characterize the influence of these residues on its enzymatic and insecticidal activities. The data gathered deepened our knowledge on ureases and will help in the future development of biotechnological applications using these proteins (or their isolated domains)

for plant protection against pests and pathogens. C. ensiformis urease Type III was purchased from Sigma–Aldrich. An Clomifene additional step of gel-filtration in a Superdex 200 Column (GE Healthcare), equilibrated in 50 mM HEPES, 250 mM NaCl, pH 7.5, was used to obtain the protein in homogeneity conditions. The purity of JBU was checked by SDS-PAGE electrophoresis. Protein content of samples was determined by their absorbance at 280 nm (A280). The extinction coefficient value (ɛ280 = 54,780 M−1 cm−1) was calculated using the ProtParam tool (http://au.expasy.org/tools/protparam.html). The methods of Hoare and Koshland (1966) and Pho et al. (1977), were followed with few adaptations. Urease (1 mg/mL), in 200 mM phosphate buffer, pH 7.0, was mixed with a solution of ethylenediamine, in phosphate buffer, with the pH previously adjusted to 7.0 using phosphoric acid.