Figure 3.

Collagenolytic and gelatinolytic activities of cathepsin K, cathepsin L, and Leu67Tyr/Ala205Leu cathepsin L mutant. (A) Cathepsins K, L, and L67Y/A205L cathepsin L mutant (600 nM) were incubated with skin collagen I (0.4 mg/mL) in 100 mM sodium acetate buffer (pH 5.5) containing 2 mM dithiothreitol and 2 mM EDTA, for 8 h at 28°C, in the presence or absence of 0.15% (w/v) C-4S. Samples were further analyzed by SDS-polyacrylamide electrophoresis using 4%–20% Tris/glycine gels (Coomassie-blue staining). Undigested bovine type I collagen was used as a standard. Molecular mass standards are indicated in the left lane. (B) Collagenolytic assays with soluble collagen II from articular joints (0.6 mg/mL).(C) Heat-denatured collagen I (gelatin; 0.4 mg/mL) was incubated with cathepsins K, L, and the two S2 mutants (10 nM), respectively, for 30 min at 28°C.