, 2004). Table 1 show that only collagenase and aminopeptidase have significant activities in salivary glands
in comparison with midgut activities, as they amount to 8–10% of the latter. Amylase and membrane-bound α-glucosidase predominate in the anterior midgut, whereas cathepsin L and collagenase are observed only in middle and posterior midguts and soluble α-glucosidase occurs along the whole midgut (Fig. 3). The supernatant obtained by centrifuging midgut homogenates of P. nigrispinus was adjusted to become 20 mM Tris–HCl buffer pH 7.0 with 1 mM MMTS and loaded onto a HiTrap Q XL column and eluted with the same buffer. Two cathepsin L-like proteinase activity peaks were observed ( Fig. 4A): CAL1, the
minor peak amounting to about 15% of midgut see more cathepsin L activity and CAL2, summing up 85% of cathepsin L activity. They were separately pooled and subsequently loaded on gel filtration columns ( Fig. 4B and C). The effect of pH (Fig. 4D) and substrate concentration (Fig. 4F) on the activity of semi-purified CAL1 were studied and the results displayed in Table 2. The same was done with CAL 2 (Fig. 4E and G, Table 2). Amylase, aminopeptidase, and soluble α-glucosidase resulted in a single activity peak FK506 after ion-exchange chromatography. Pooled fractions corresponding to each enzyme were thereafter submitted to gel filtration, resulting again in single activity peaks (not showed). The pH optima, molecular masses and km values of the semi-purified enzymes are displayed in Table 2. Two α-glucosidases were found in P. nigrispinus midguts: one soluble and another membrane bound. The latter should correspond to the enzyme marker of the perimicrovillar membranes found in hemipterans and insects pertaining to some other paraneopteran P-type ATPase orders ( Terra and Ferreira, 1994, Terra and Ferreira, 2012 and Silva et al., 2004). There is a single molecular
species of the soluble α-glucosidase, amylase, and aminopeptidase, which have properties similar to those described from other insects, including hemipterans ( Terra and Ferreira, 1994 and Terra and Ferreira, 2012). In D. peruvianus, a Hemiptera Pentatomomorpha like P. nigrispinus, the aminopeptidase is found in the space between the microvillar and perimicrovillar membranes, where it carries out the intermediate digestion of proteins ( Silva et al., 1996). Cathepsin Ls are major digestive proteinases in Cucujiformia beetles and in hemipterans. The digestive enzymes were derived from an ancestral gene that codes for a lysosomal cathepsin L. Digestive beetle cathepsin L seem to be more derived (farther from the lysosomal enzyme) than those from hemipterans (Terra and Ferreira, 2012). P. nigrispinus is not an exception among hemipterans, as no serine proteinases (chymotrypsin and trypsin) were found in their midguts.
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