The crude extract of whole midgut S. levis larvae was submitted to ion exchange chromatography in DEAE-Sepharose. A large peak of inactive protein was eluted with 0.3 M NaCl. Two other peaks were eluted selleck compound in 1 M NaCl ( Fig. 4A). These two peaks hydrolyze Z-FR-MCA, but most of the activity was associated with the second peak. SDS-PAGE of the purified proteins
revealed a single band corresponding to each eluted peak, displaying the same molecular mass of approximately 37 kDa ( Fig. 4B). As the enzyme present in the second peak has greater activity and was more stable than the first, it was chosen for characterization. Thus, the data refer only to the major S. levis midgut cathepsin L. The successfully purified enzyme is active on Z-FR-MCA, has an optimal pH of 6 (Fig. 5). The kinetic parameters for the hydrolysis of the fluorogenic peptides Z-FR-MCA, Z-RR-MCA and Z-LR-MCA by S. levis cysteine proteinase were determined. The greatest catalytic efficiency was obtained with Z-FR-MCA with kcat/Km value of 30.0 ± 0.5 μM−1 s−1. The substrate Z-LR-MCA was hydrolyzed with a kcat/Km value of 20.0 ± 1.1 μM−1 s−1 and Z-RR-MCA substrate was resistant to hydrolysis. The kinetic data and standard deviations were calculated from at least three separate determinations. Amylase and maltase were assayed throughout the midgut to
define the sites of initial (amylase) and final (maltase) starch digestion. Cysteine proteinase selleck chemicals and trypsin were found to be the major and minor digestive proteinases, respectively,
in S. levis (see previous item). Hence, both proteinase activities were selected NADPH-cytochrome-c2 reductase to identify the site of initial protein digestion and that of final digestion of aminopeptidase. Optimal pH for the selected enzymes are ( Fig. 5) 6–7 for amylase, 5–6 for maltase, 8–10 for trypsin, 7–8 for aminopeptidase and 6.0 for cysteine proteinase. The selected enzymes were analyzed in the midgut contents and in the soluble and membrane-bound fraction of the midgut tissue at different sites along the midgut ( Fig. 6). Based on the data, amylase, maltase, cysteine proteinase and trypsin predominate in the luminal contents of the anterior (V1 and V2) midgut. However, trypsin also occurs in significant amounts in the tissue both as a soluble and as a membrane-bound enzyme ( Fig. 6). An aminopeptidase is found mainly in the posterior (V3 + V4) midgut as a membrane-bound enzyme ( Fig. 6). The midgut of S. levis has two cysteine proteinases, two trypsins and perhaps a negligible chymotrypsin. SDS-PAGE analysis showed purified bands of cysteine proteinases both with 37 kDa eluted at 1 M NaCl as two peaks. This elution profile suggests the presence of two isoforms of cysteine proteinase that most likely differ in their charge or isoeletric point. S. levis cathepsin L exhibits elution profile similar to human cathepsin L (EC 22.214.171.124) purified from human kidneys ( Turk, 1993). The major S.