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. Author manuscript; available in PMC: 2010 Oct 30.
Published in final edited form as: Chem Biol. 2009 Oct 30;16(10):1053–1063. doi: 10.1016/j.chembiol.2009.09.009

Figure 2. Imaging of Tick Gut Peptidases with Active-Site Probes.

Figure 2

Extract from the gut tissue of I. ricinus was treated with a panel of selective activity-based probes (ABPs) that interact with the active site of the target peptidases.

(A) ABPs for labeling of cysteine peptidases: Green-DCG-04 for cathepsin B (CatB) and cathepsin L (CatL), FY01 for cathepsin C (CatC), and Fhex-PD-AOMK for legumain (AE). The ABP structures contain an irreversible active-site ligand as chemically reactive warhead (gray box) and fluorescent tag as a reporter group (open oval).

(B) ABP named bPSP-07 for labeling of aspartic peptidase cathepsin D (CatD). The bPSP-07 structure contains a reversible active-site ligand (gray box), which is cross-linked to the protein target by a photoreactive group, and biotin tag as a reporter group (open oval).

The reaction mixture was resolved by Laemmli-SDS-PAGE and the labeled peptidases were visualized in the gel (fluorescence signal) or on the chemiluminescent avidin blot (biotin signal). The competitive labeling was performed in the presence of selective active-site inhibitors: CA-074 (CatB inhibitor), Z-Phe-Phe-DMK (CatL inhibitor), Aza-N-11a (AE inhibitor), Gly-Phe-DMK (CatC inhibitor) and pepstatin (CatD inhibitor) (for details, see Experimental procedures).