H gly phe 7 amino 4 methylcoumarin amc

Enzyme activities were determined using specific fluorogenic substrates: 70 μM H-Gly-Phe-7-amino-4-methylcoumarin (AMC) (Bachem) for cathepsin C, 20 μM H-Arg-AMC (Bachem) for cathepsin H, 50 μM Z-Phe–Arg-AMC for cathepsin L (Bachem) and 50 μM acetyl-Ile-Glu-Pro-Asp-AMC for granzyme B (Bachem). The assay buffers used were 25 mM MES, 100 mM NaCl, 5 mM cysteine, pH 6 for cathepsin C, 100 mM MES, 2mM EDTA, 5 mM cysteine, pH 6.5 for cathepsins H and L and 50 mM Tris-HCl, 100 mM NaCl, pH 7.4 for granzyme B. Whole-cell lysates were first activated in assay buffer for 15 minutes at room temperature for cathepsins or for 30 minutes at 37°C for granzyme B. The substrate was then added and formation of fluorescent degradation products was measured continuously with excitation at 370 nm and emission at 460 nm on a microplate reader Infinite M1000 (Tecan, Männedorf, Switzerland). To determine cathepsin L activity, 5 μM irreversible inhibitor of cathepsin B, CA-074 (Bachem), was added before the addition of substrate. The rate of AMC release was calculated and normalised to the enzyme protein levels determined from western blot. The activity of the control sample was set to 100% and activities of other samples were adjusted accordingly.