Figure 1

Characterization of various cathepsins and their inhibition by cysC in vitro . (A) Evaluation of cathepsins in activity assay. Cleavage of 10 μM substrate by 30 nM of each cathepsin (catB, catK, catL, catS and catH) at 30 min was measured by RFUs as described in Methods. Values represent means for each condition (n = 2). A representative of two independent experiments is shown. Insert shows EC₅₀ for each cathepsin tested. Mean EC₅₀ = 21 nM ± 4 for catK was based on four independent experiments. (B) Inhibition of cathepsin activity by cysC. Assays were performed using 25 nM of cathepsin and 50 μM of substrate in two to three independent experiments and the values represent means for each condition (n = 2–3). The data are shown as percentage of inhibition of activity compared no cysC present. Insert shows IC₅₀ values at conditions used. (C) Inhibition of human catK by cysC. The potency of mouse and human cysC on catK activity (60 nM) with 50 μM of substrate was compared and inhibition was measured as RFUs at 20 min. IC₅₀ was 65 nM for both cysC proteins. cat, cathepsin; cysC, cystatin C; RFU, relative fluorescence units.