Microplate Assay For Cathepsin Detection in Viable Cells Using Derivatives of 4‐Methoxy‐β‐Naphthylamide

Anke Ruettger1, Bernd Wiederanders2

1 Orthopedical Research Unit Eisenberg, Universitätsklinikum, Friedrich‐Schiller‐Universität Jena, 2 Institute of Biochemistry I, Universitätsklinikum, Friedrich‐Schiller‐Universität Jena
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.21
DOI:  10.1002/0471140864.ps2121s49
Online Posting Date:  August, 2007
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Abstract

This unit describes an assay for the direct and selective detection of the four cathepsins B, H, K, and L in adherently growing cells. Cells are incubated with substrates that are peptidic derivatives of 4‐methoxy‐β‐naphthylamine partially selective for each cathepsin, together with 5‐nitrosalicylaldehyde. The protease reaction is performed in microtiter plates and the fluorescent hydrolysis products are detected using a plate reader. The selectivity of detection is enhanced by parallel assays containing inhibitors that are also partially selective for each of the cathepsins. Individual cathepsin activities can then be calculated by the difference between the uninhibited and the inhibited assays. Detection of cathepsin H activity differs from the other assays in that other nonlysosomal aminopeptidases are inhibited by bestatin. The most common application of these assays is to compare directly cells treated with different substances, e.g., pharmaceutically interesting cathepsin inhibitors. Curr. Protoc. Protein Sci. 49:21.21.1‐21.21.8. © 2007 by John Wiley & Sons, Inc.

Keywords: in vivo cathepsin assay; live cells; selectivity; microplate assay

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cells
  • 5‐nitrosalicylaldehyde solution (see recipe)
  • Substrate solutions (see reciperecipes)
  • Inhibitor solutions (see reciperecipes)
  • Phosphate‐buffered saline (PBS; see recipe), prewarmed
  • 96‐well Black/Clear Assay Plates (Optilux, BD Biosciences)
  • Multichannel pipettor
  • Microtiter plate reader with fluorescence detection (e.g., Fluor‐S Multi‐Imager from Bio‐Rad)
  • Antifade Kit (ProLong, Molecular Probes Invitrogen detection technologies, P7481), optional
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Figures

Videos

Literature Cited

Literature Cited
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   Bromme, D., Okamoto, K., Wang, B.B., and Biroc, S. 1996. Human cathepsin O2, a matrix protein‐degrading cysteine protease expressed in osteoclasts. Functional expression of human cathepsin O2 in Spodoptera frugiperda and characterization of the enzyme. J. Biol. Chem. 271:2126‐2132.
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   Rawlings, N.D., Morton, F.R., and Barrett, A.J. 2006. MEROPS: the peptidase database. Nucleic Acids Res. 34:D270‐272.
   Rothe, G., Klingel, S., Assfalg‐Machleidt, I., Machleidt, W., Zirkelbach, C., Banati, R.B., Mangel, W.F., and Valet, G. 1992. Flow cytometric analysis of protease activities in vital cells. Biol. Chem. Hoppe Seyler 373:547‐554.
   Ruettger, A., Mollenhauer, J., Loeser, R., Guetschow, M., and Wiederanders, B. 2006. Microplate assay for quantitative determination of cathepsin activities in viable cells using derivatives of 4‐methoxy‐β‐naphthylamide. Biotechniques 41:469‐472.
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   Umezawa, H., Aoyagi, T., Suda, H., Hamada, M., and Takeuchi, T. 1976. Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes. J. Antibiot. (Tokyo) 29:97‐99.
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