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Measuring Cysteine Cathepsin Activity to Detect Lysosomal Membrane Permeabilization
Urška Repnik1, Maruša Hafner Česen2, Boris Turk3
1Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, SI-1000 Ljubljana, Slovenia; Department of Biosciences, University of Oslo, NO-0371 Oslo, Norway;
Cold Spring Harbor Protocols
|May 4, 2016
Summary
This study presents a new protocol to measure cysteine cathepsin activity, a key indicator of lysosomal membrane permeabilization (LMP). The method uses digitonin to permeabilize cell membranes, enabling sensitive and high-throughput detection of LMP.
Area of Science:
- Cell Biology
- Biochemistry
- Enzymology
Background:
- Lysosomal membrane permeabilization (LMP) releases cellular contents into the cytosol.
- Cysteine cathepsins are small lysosomal enzymes, making their activity a reliable marker for LMP.
- Existing methods for detecting LMP can be improved for sensitivity and throughput.
Purpose of the Study:
- To develop and present a protocol for measuring cysteine cathepsin activity as a hallmark of LMP.
- To enable quantification of lysosomal enzyme release after selective or total cell membrane permeabilization.
- To provide a sensitive, high-throughput method applicable to various cell lines.
Main Methods:
- Selective plasma membrane permeabilization using low-concentration digitonin.
- Total cell membrane lysis using high-concentration digitonin.
- Measurement of cysteine cathepsin activity using fluorogenic substrates in lysed cells or cell-free extracts.
Main Results:
- The protocol allows for the measurement of cysteine cathepsin activity in both lysed cells and cell-free extracts.
- The method is sensitive and suitable for high-throughput screening.
- The protocol demonstrates general applicability across different cell lines.
Conclusions:
- This protocol offers a robust method for assessing lysosomal membrane permeabilization by measuring cysteine cathepsin activity.
- The technique is adaptable for different experimental needs, whether examining intact lysed cells or cell-free extracts.
- The developed method enhances the study of LMP in various biological contexts.
