The activities of the α-amylase and α-glucosidase were assayed using starch and p-Np-α-d-glucopyranoside as substrates, respectively (Sections 2.2.1 and 2.3.1). The column was calibrated with BSA (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa). The molecular mass of the α-amylase was also evaluated using SDS–PAGE. Twenty midguts were homogenized in 20 μL of 0.9% (w/v) saline and centrifuged at 14,000×g for 10 min at 4 °C. The supernatant was mixed with 20 μL of the sample buffer

(2 X concentrated, without mercaptoethanol) and was not heated. Pre-stained proteins were used as molecular TGF-beta inhibitor mass standards (Thermo Scientific code 26612). The electrophoresis was performed in a polyacrylamide gel (10%) at room temperature and a constant voltage

of 100 V according to the method of Laemmli (1970). Following the electrophoresis, the gel was washed in an aqueous solution of 2.5% (v/v) Triton X-100 for 1 h at room temperature and placed under a second gel that was copolymerized with 0.5% soluble starch and 0.05 M HEPES buffer pH 8.5 containing 20 mM NaCl. The gels were then placed in a semidry system between sheets of filter paper that were previously soaked in buffer. After incubation at 30 °C for 12 h, the bands were revealed by treatment with Lugol (0.5% I2 and 1% KI). The determination of the protein concentration this website was achieved by the BCA methodology (BCA Protein Assay – Pierce) (Stoscheck, 1990). One unit (U) of enzyme

activity was defined as the amount of enzyme capable of producing 1 μmol of product.min−1 under the assay conditions. A photograph of the digestive tube of the L. longipalpis fourth instar larvae is presented in Fig. 1. According to our results, the amylolytic activity is maximal at pH 8.5 ( Fig. 2) and can be observed throughout the midgut; this activity predominates Methocarbamol in the anterior midgut, where approximately 2/3 of all the activity is concentrated ( Fig. 3(a). A similar pattern was observed using glycogen as a substrate (data not shown). All of the amylolytic activity measured in the present article can be attributed to the larvae; whereas the amylolytic activity of the larvae is higher at pH 8.5 (its optimum pH), that of the fungi obtained from the rearing pots is higher at pH 6.5. Two soluble enzymes were responsible for the amylolytic activity observed in the midgut of the larvae ( Fig. 3(b) and Fig. 4(a). The apparent molecular masses of these two enzymes were 103 and 45 kDa. It was not possible to determine the molecular mass of the α-amylase using gel filtration because of a non-sieving interaction between the enzyme and the resin used for the chromatography. The optimum pH for α-amylase activity (pH 8.5) is in accordance with the pH observed in the lumen of the anterior midgut (Fig. 1), the site where the enzymes predominates (Fig. 3(a).