All assays were performed under conditions in which the product was proportional to enzyme concentration and incubation this website time. Controls without enzyme and others without substrate were included. One general proteinase unit is the amount of enzyme that causes an increase in the emission of 1000 units/60 min. For the other enzymes, one enzyme unit is the amount that hydrolyzes 1 μmol of substrate (or bond) per min. Enzyme activity is expressed in milli units (mU). Ten
S. levis larvae were maintained at 4 °C for 5 min, dissected and the whole midgut were homogenized in buffer containing Tris–HCl 10 mM, NaCl 150 mM and 2% Triton X-100, pH 7.4 (2 ml). The mixture was centrifuged at 6000 × g for 30 min. The soluble fraction was applied
to a DEAE-Sephadex column (25 cm × 1 cm) equilibrated with 0.1 M Tris–HCl, pH 8.0. The proteins were eluted with 1.0 M NaCl in the same buffer. The protein elution profile was followed by UV absorbance (280 nm). After protein elution, dialysis was performed in a buffer containing 10 mM Tris–HCl and 50 mM NaCl, pH 8.0. The hydrolysis of the fluorogenic peptides Z-FR-MCA, Z-LR-MCA and Z-RR-MCA (Calbiochem, La Jolla, CA, USA) by purified S. levis peptidase was continuously monitored in a Hitachi F-2500 spectrofluorimeter by measuring fluorescence at λex = 380 nm and λem = 460 nm. Approximately 20 μM of purified enzyme were added to 0.1 M sodium acetate, selleck products pH 5.5, containing 2.5 mM DTT (1.0 ml final volume) and incubated for 3 min at 37 °C. The substrates were then added at different concentrations and the catalytic activity was monitored. The apparent second-order rate constant Kcat/Km was determined under pseudo first-order conditions, in which [S] ≪ Km. Determinations were performed with different substrate concentrations and calculated using nonlinear regression
data analysis with the aid of the GraFit program ( Leatherbarrow, 2001). The molar concentration of the S. levis cysteine proteinase was determined by active site titration with E-64 inhibitor ( Anastasi et al., 1983). The pH dependence on Z-FR-MCA hydrolysis by S. levis proteinase Cobimetinib price was studied over a range of 4.0–9.0. Determinations were carried out at 37 °C using the following buffers: 0.1 M sodium acetate (4.0 < pH < 5.5); 0.1 M sodium phosphate (6.0 < pH < 7.0); 0.1 M Tris–HCl (7.0 < pH < 8.5) and 0.1 M sodium borate (9.0 < pH < 10.0). The enzyme was pre-activated with 2.5 mM DTT for 5 min at 37 °C before the addition of the substrate. Enzyme activity was monitored using the fluorimetric assay described above. For each pH value, enzyme activity was calculated using the Grafit program ( Leatherbarrow, 2001). All experiments were carried out in triplicate and the values were converted to percentage of relative activity. The gut of the larvae is composed of a very short foregut, a large midgut that is anteriorly dilated and a medium-size hindgut (Fig. 1). The midgut is made up of a simple linear tube – ventriculus.
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