FIGURE 5.

HPLC analysis of endosomal reaction mixtures treated with the CatS inhibitor LHVS. Chromatograms of three substrates PMGLP (A), PMGAP (B), and KVSVR (C) at a concentration of 20 μm after incubation with endosomal fractions from macrophages (0.67 μg of total protein) for 2 h in digestion buffer at pH 5.5 and 37 °C. Endosomal fractions were preincubated for 1 h at 37 °C: Left column, without inhibitor; right column, with 10 nm LHVS. Digestion products were detected by their UV absorbance at 220 nm. The black lines represent chromatograms of the digestion mixtures, whereas the gray lines are chromatograms of the undigested negative controls. LHVS prevents the digestion of the two substrates PMGLP and PMGAP, whereas the unspecific substrate KVSVR is still digested under similar conditions.