Figure 5
Catalytic activity of ferret anionic and cationic trypsins. (A) Enzyme kinetic parameters determined on the N-CBZ-Gly-Pro-Arg-p-nitroanilide substrate at 22 °C. Incubations were performed in 0.1 M Tris-HCl (pH 8.0), 1 mM CalCl2 and 0.05% Tween 20 with 1 nM trypsin (final concentrations). (B) Activation of bovine chymotrypsinogen (2 µM) with 25 nM ferret trypsins at 37 °C in 0.1 M Tris-HCl (pH 8.0), 1 mM CalCl2 and 0.05% Tween 20 (final concentrations). At the indicated times 2 µL aliquots were withdrawn and chymotrypsin activity was measured with the Suc-Ala-Ala-Pro-Phe-p-nitroanilide substrate. Mean values with S.D. are shown. (C) Digestion of bovine β-casein with ferret trypsins. Casein (0.2 mg/mL) was incubated with ferret trypsin (5 nM) in 0.1 M Tris·HCl (pH 8.0) and 1 mM CaCl2 (final concentrations) at 37 °C. At the indicated times, 75 µL aliquots were precipitated with 10% trichloroacetic acid, electrophoresed on 15% SDS-PAGE minigels, and stained with Coomassie Blue. A representative gel from two experiments is shown.
