Fluorometric Microassays for the Determination of Cathepsin L and Cathepsin S Activities in Tissue Extracts
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Bernd Werle
Abstract
We established a continuous semi-microassay, and for large-scale studies both a stopped and a continuous microtiter plate assay for the fluorometric determination 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 cathepsin B the specific inhibitor CA-074 for blocking interfering cathepsin B activities was applied. Furthermore, 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 semimicroassay were 8.1%–8.9%, while for the stopped and continuous microtiter plate assays the coefficients were 11.2%–13.5% and 5.8%–12.2%, respectively. Compared to the continuous semi-microassay, the stopped and the continuous microtiter plate assays showed 3-fold and 11-fold higher sensitivity, respectively. Comparison between the continuous enzyme activity assays at substrate concentrations of 40 μM and 200μM demonstrated a significant correlation of r = 0.97 and r = 0.99, respectively.
The newly developed microtiter plate assay will allow efficient, sensitive and high precision determination of cathepsin L and cathepsin S activities in large-scale studies of cysteine-cathepsin dependent diseases.
Copyright (c) 1999 by Walter de Gruyter GmbH & Co. KG
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- Two Highly Related Homeodomain Proteins, Nkx5-1 and Nkx5-2, Display Different DNA Binding Specificities
- The Janus Face of the Archaeal Cdc48/p97 Homologue VAT: Protein Folding versus Unfolding
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- On the Lysosomal Degradation of Neurofibromin and Its Phosphorylation in Cultured Melanocytes
- Rat Muscle Fructose-1,6-Bisphosphatase: Cloning of the cDNA, Expression of the Recombinant Enzyme, and Expression Analysis in Different Tissues
- Catalase-Peroxidase from the Cyanobacterium Synechocystis PCC 6803: Cloning, Overexpression in Escherichia coli, and Kinetic Characterization
- Expression of Plasma Prekallikrein mRNA in Human Nonhepatic Tissues and Cell Lineages Suggests Special Local Functions of the Enzyme
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