An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells
| Autor: | Paul Tawa, M. David Percival, Wanda Cromlish, Kathryn E. Bass, Sylvie Desmarais, Shankar Venkatraman, Sonia Lamontagne, W. Cameron Black, Gregg Wesolowski, Jean-Pierre Falgueyret, Sevgi B. Rodan, Denis Riendeau, Christophe Mellon |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Cathepsin L
Blotting Western Cathepsin K Biophysics Cathepsin E Cathepsin F Cysteine Proteinase Inhibitors Biology Biochemistry Cathepsin A Cathepsin B Cathepsin C Iodine Radioisotopes Cathepsin O Leucine Cathepsin H Cell Line Tumor Cathepsin L1 Animals Humans Molecular Biology Biphenyl Compounds Cell Biology Cathepsins Molecular biology Cysteine Endopeptidases Diazomethane Autoradiography Rabbits |
| Zdroj: | Analytical Biochemistry. 335:218-227 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2004.09.005 |
| Popis: | We describe a novel diazomethylketone-containing irreversible inhibitor (BIL-DMK) which is specific for a subset of pharmaceutically important cysteine cathepsin proteases. BIL-DMK rapidly inactivates cathepsins B, F, K, L, S, and V in isolated enzyme assays and labels cathepsins in whole cells. The presence of catalytically active cathepsins B, L, and K or S was demonstrated using radioiodinated BIL-DMK in HepG2 (hepatoma), HIG82 (rabbit synoviocyte), and Ramos (B lymphoma) cell lines, respectively. The identity of each protein labeled was confirmed from the isoelectric point and molecular mass of the radioactive spots on two-dimensional gel and by comigration with each cathepsin as identified by immunoblotting. These cell lines were used to establish whole-cell enzyme occupancy assays to determine the potency of both irreversible and reversible inhibitors against each cathepsin in their native cellular lysosomal or endosomal environment. These whole-cell enzyme occupancy assays are useful to determine the cellular permeability of competing inhibitors and have the advantage of not requiring specific substrates for each cathepsin of interest. |
| Databáze: | OpenAIRE |
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