Biol. Chem., Vol. 380, pp. 1109 –1116, September 1999 · Copyright © by Walter de Gruyter · Berlin · New York Fluorometric Microassays for the Determination of Cathepsin L and Cathepsin S Activities in Tissue Extracts Bernd Werle 1,2, *, Alexander Staib 1 , Britta Jülke 1 , Werner Ebert 1 , Pavel Zladoidsky 3 , Andreja Sekirnik 4 , Janko Kos 4,5 and Eberhard Spiess 2 1 Thoraxhospital Heidelberg-Rohrbach, Amalienstr. 5, D-69126 Heidelberg, Germany 2 Deutsches Krebsforschungszentrum, Biomedizinische Strukturforschung, Im Neuenheimer Feld 280, D-69120, Heidelberg, Germany 3 Drug Research Institute, 90001 Modra, Slovakia 4 Jozef-Stefan-Institute, SI-61111 Ljubljana, Slovenia 5 KRKA d.d., R&D Division, Department of Biochemical Research and Drug Design, Novo Mesto, SI-8000 Ljubljana, Slovenia * Corresponding author We established a continuous semi-microassay, and for large-scale studies both a stopped and a continu- ous microtiter plate assay for the fluorometric deter- mination of cathepsin L and cathepsin S activities in body fluids, tissues or cell extracts in the presence of cathepsin B. For the detection of enzymatic activities we used the synthetic substrate Z-Phe-Arg-AMC, and for discrimination between cathepsin L, S and cathep- sin B the specific inhibitor CA-074 for blocking inter- fering cathepsin B activities was applied. Further- more, we took advantage of the stability of cathepsin S at pH 7.5 for further differentiation between cathepsin L and cathepsin S activities. The kinetic assays were characterized in terms of imprecision, analytical sensitivity, accuracy and substrate concentration. The within-run coefficients of variation were found to be 4.9% –7.2% for the continuous semi-microassay, 10.3% –11.7% for the stopped, and 4.5% –11.8% for the continuous microtiter plate assay. The between- days coefficients of variation for the continuous semi- microassay were 8.1% –8.9%, while for the stopped and continuous microtiter plate assays the coeffi- cients were 11.2% –13.5% and 5.8% –12.2%, respec- tively. Compared to the continuous semi-microassay, the stopped and the continuous microtiter plate as- says showed 3-fold and 11-fold higher sensitivity, re- spectively. Comparison between the continuous en- zyme activity assays at substrate concentrations of 40 M and 200 M demonstrated a significant correla- tion of r = 0.97 and r = 0.99, respectively. The newly developed microtiter plate assay will allow efficient, sensitive and high precision determi- nation of cathepsin L and cathepsin S activities in large-scale studies of cysteine-cathepsin dependent diseases. Key words: Cathepsin S / Cysteine cathepsins / Kinetic assay / Lung cancer. Introduction Cysteine peptidases (CPs) such as cathepsin B (cath B; EC 3.4.22.1), cathepsin H (cath H; EC 3.4.22.16), and cathepsin L-type enzymes, i. e. cathepsin L (cath L; EC 3.4.22.15) and cathepsin S (EC 3.4.22.27), have important functions not only in protein catabolism but also in tissue remodeling, hormone activation and antigen presentation (reviewed by Kirschke et al., 1995). In addition, these en- zymes may also be involved in diseases such as arthritis, Alzheimer’s disease, osteoporosis, lung disorders and tu- mor invasion and metastasis (Burnett and Stockely, 1985; Berquin et al., 1994; Kirschke et al., 1995; Elliott et al., 1996). To establish the specific role of the various cathep- sins in pathobiochemistry it is necessary to assess their activity levels. Up to now selective assessment is only possible for cath B and cath H by using the synthetic specific substrates 7-(N-benzyloxycarbonyl-L-arginyl-L- arginylamido)-4-methylcoumarin · HCl · H 2 O (Z-Arg-Arg- AMC) and 7-(L-arginylamido)-4-methylcoumarin (H-Arg- AMC), respectively (Barrett and Kirschke, 1981). In con- trast, there is no synthetic specific substrate available for cath L and/or cath S. The favorite substrate for cath L- type enzymes, 7-(N-benzyloxycarbonyl-L-phenylalanyl- L-arginylamido)-4-methylcoumarin (Z-Phe-Arg-AMC), is also cleaved by cath B, particularly when cath B is present in excess over cath L and S (Barrett and Kirschke, 1981; Lesser et al., 1989; Lah et al., 1992; Werle et al., 1995; Ulbricht et al., 1996). To delimit cath L-type activities from cath B activities one of these enzymes has to be selective- ly blocked. Several authors reported on the discrimination of cath L-type activities and cath B activities by using the inhibitor N-benzyloxycarbonyl-L-phenylalanyl-L-phenyl- alanyl-diazomethylketon (Z-Phe-Phe-CHN 2 ) (Barrett and Kirschke, 1981; Lesser et al., 1989; Lah et al., 1992; Werle et al., 1995). However, this approach is not reliable for the determination of small amounts of cath L-type activities in the presence of an excess of cath B activity as we showed earlier (Werle et al., 1995). The application of N-(L-3- trans- propyl-carbamoyl-oxirane-2-carbonyl)-L-isoleucyl-L-pro- Brought to you by | Universität Osnabrück Authenticated Download Date | 5/27/15 2:02 PM