FIGURE 4.

Specific inhibition of CatS prevents digestion of designed substrates by endosomal fractions; comparison with an unspecific substrate. Time-progress curves of peptide substrate hydrolysis reactions catalyzed by macrophages endosomal fractions followed by fluorescence emission. 0.67 μgof total protein from macrophage endosomal fractions were preincubated for 1 h at 37 °C: ▪, without inhibitor; , with 10 nm LHVS. All of the assays were performed in triplicate at 37 °C in 50 mm sodium acetate buffer (pH 5.5). A, hydrolysis of the internally quenched fluorescent peptide Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH₂ (PMGLP) at a concentration of 20 μm. Progress of product formation was recorded over 2 h by fluorescence emission of the N-terminal Mca group at 405 nm, following extinction at 340 nm. LHVS prevents the hydrolysis. B, hydrolysis of the internally quenched fluorescent peptide Mca-GRWHPMGAPWE-Lys(Dnp)-DArg-NH₂ (PMGAP) at a concentration of 20 μm. Progress of product formation was recorded over 3 h (λ_ex = 340 nm, λ_em = 405 nm). LHVS prevents the hydrolysis. C, hydrolysis of the unspecific fluorogenic peptide Z-FR-AMC at a concentration of 100μm. Progress of product formation was recorded over 2 h by fluorescence emission of the AMC group (λ_ex = 360 nm, λ_em = 465 nm). LHVS reduces the initial velocity of the hydrolysis reaction but does not prevent it.