title: Measuring Cysteine Cathepsin Activity to Detect Lysosomal Membrane Permeabilization.
abstract: During lysosomal membrane permeabilization (LMP), lysosomal lumenal contents can be released into the cytosol. Small molecules are more likely to be released, and cysteine cathepsins, with mature forms possessing a mass of 25-30 kDa, are among the smallest lumenal lysosomal enzymes. In addition, specific substrates for cysteine cathepsins are available to investigators, and therefore the measurement of the cathepsin activity as a hallmark of LMP works well. Here, we present a protocol for measuring the activity of these enzymes after selective plasma membrane permeabilization with a low concentration of digitonin and after total cell membrane lysis with a high concentration of digitonin. A fluorogenic substrate can be added either directly to the well with lysed cells to show LMP or to the cell-free extract to show that the lysosomal membrane has been sufficiently destabilized to allow the translocation of lysosomal enzymes. Although the content of lysosomal cysteine cathepsins differs between cell lines, this method has general applicability, is sensitive, and has high throughput. The presented protocol shows how to measure cysteine cathepsin activity in the presence of lysed cells and also in cell-free extracts. Depending on the aim of the study, one or both types of measurements can be performed.
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, SI-1000 Ljubljana, Slovenia; Department of Biosciences, University of Oslo, NO-0371 Oslo, Norway;
authors.forename: Urška
authors.initials: U
authors.lastname: Repnik
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, SI-1000 Ljubljana, Slovenia;
authors.forename: Maruša Hafner
authors.initials: MH
authors.lastname: Česen
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, SI-1000 Ljubljana, Slovenia; Center of Excellence CIPKEBIP, SI-1000 Ljubljana, Slovenia.
authors.forename: Boris
authors.initials: B
authors.lastname: Turk
mesh_terms: D002403:Cathepsins / Q000032:analysis*; D003584:Cytological Techniques / Q000379:methods*; D007425:Intracellular Membranes / Q000502:physiology*; D008247:Lysosomes / Q000201:enzymology*; D008566:Membranes; D010539:Permeability
chemical_list: D002403:Cathepsins