An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells
Section snippets
Methods
Recombinant rabbit Cat K and human Cat F, L, S, and V were prepared as previously described [9], [12], [13]. Human liver Cat B was from Sigma, while recombinant human Cat C and Z were from R&D Systems. Cathepsin H was from Biomol. All protease substrates were from Bachem. Cbz-Y-A-DMK was from Enzyme Systems Probes and was radioiodinated by Amersham Pharmacia Biotech or with nonradioactive iodine using NaI and chloramine-T in dimethylformamide [14]. Dipeptidyl nitrile A was prepared by the
Inactivation of cathepsin activities by BIL-DMK
The irreversible inhibitor Cbz-Y(I)-A-DMK is a potent inhibitor of cathepsin L and V [1] but shows significantly weaker inhibition of cathepsins B, F, S, and K (Table 1). When cells from the human hepatoma cell line HepG2 were treated for 60 min with 5 nM radioiodinated form of Cbz-Y(I)-A-DMK and the samples analyzed by 2D gel electrophoresis and autoradiography, labeling of Cat B and L was readily detected (results not shown). In contrast, a relatively low amount of Cat S labeling was observed
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