The tubes were incubated at 37 °C for 15 min, and the absorption at 505 nm was measured in a cuvette. The following buffers were used in the assays: 0.1 M acetic acid/NaOH MK-2206 research buy (pH 4.5, 5.0 or 5.5),

0.1 M MES/NaOH (pH 6.0, 6.5 or 7.0) and 0.1 M HEPES/NaOH (pH 7.5, 8.0 or 8.5). The blanks were prepared using heat-inactivated samples (2 min in boiling water). Isomaltose was assayed at pH 6.5 using this protocol. For calculations, a standard curve was obtained with different quantities of glucose dissolved in 10 μL of water and reacted with 1 mL of PAP reagent according the method above described. Five insects were dissected in 0.9% (w/v) NaCl. Each intestine was cut into four pieces (anterior midgut, middle midgut, posterior midgut

and hindgut), which were transferred to four different micro centrifuge tubes containing 500 μL of 0.9% (w/v) NaCl and 1% (v/v) Triton X-100. After homogenization, the tubes were centrifuged at 14,000×g for 10 min at 4 °C, and the supernatant was used in the assays. Maltose or trehalose were used as substrates and assayed as described in Section 2.3.2 at pH 6.5 and pH 6.0, respectively. The samples were prepared as described in Section 2.2.3 and assayed using maltose (pH 6.5) or trehalose (pH 6.0) as substrates according the methodology described in Section 2.3.2. To investigate whether the enzymes are bound to intestinal microvilli, the larval microvilli were purified according to the method of Abdul-Rauf and Ellar (1999). Sixty larvae were dissected in 0.9% saline (w/v), the luminal content Baf-A1 in vitro A-1210477 datasheet was discarded, and the midgut walls were washed and transferred to 40 μL of an ice-cold MET solution (300 mM mannitol, 5 mM EGTA, 17 mM TRIS-base/HCl, pH 7.5) in a micro centrifuge tube. The midguts were manually homogenized with an abrasive glass microhomogenizer for 15 min in an ice bath, and the volume was brought to 100 μL with the same solution. One hundred microliters

of ice-cold 24 mM MgCl2 was added to this preparation and the tube content was mixed and separated into two aliquots of 100 μL each. After 20 min on ice, one of the aliquots was centrifuged at 2500×g for 15 min at 4 °C. The supernatant was collected in another tube, and the pellet was rehomogenized in 100 μL of a fresh ice-cold MET/MgCl2 (1:1) solution and centrifuged. After repeating this procedure three times, the supernatants were mixed and centrifuged at 25,000×g for 30 min at 4 °C. The pellet, enriched with microvillosites, was dissolved in 100 μL of MET/MgCl2 (1:1) containing 1% Triton X-100 (v/v). Triton X-100 was also added to the non-centrifuged aliquot to a final concentration of 1% (v/v) and mixed. Both the centrifuged and non-centrifuged aliquots were centrifuged at 14,000×g, and the supernatants were used for the assays.