Enzymatic Activity Measurements of Lysosomal Hydrolases in Cytoplasm

Background of Enzymatic Activity Measurement of Lysosomal Hydrolases

After lysosomal membrane permeabilization (LMP), harmful hydrolyzed components in lysosomes are released into the cytosol. The degree of membrane permeabilization determines cell fate, and downstream signaling involves activation of caspases through intrinsic or extrinsic pathways to induce classical apoptosis. The ability of lysosomes to induce cell death signals makes them interesting targets in cancer therapy, especially for apoptosis and multidrug-resistant cancers. Detergents are useful molecular tools in biophysics and molecular biology, with common uses including solubilization and recombination of membrane proteins, genes and drugs, and membrane solubilization. The solubilization of lipid membranes by detergents has received great attention.

Fig. 1. Flowchart of the digitonin extraction procedure for measuring LMP. (Jäättelä M, et al., 2015)

Enzymatic Activity Measurement of Lysosomal Hydrolases Analysis Services

Traditionally lysosomal membrane permeability has been assessed using a β-glycerophosphate substrate, but this substrate readily penetrates the lysosomal membrane only under conditions of altered permeability. CD BioSciences offers multiple methods to quantify and visualize lysosomal permeability during cell death. Here, we quantified the release of cathepsin and β-N-acetylglucosaminidase into the cytoplasm by measuring the enzymatic activity to assess LMP.

Because cathepsin has a short half-life in the cytosol, the values obtained are actually lower than the actual amount of hydrolase released. We optimized a detailed protocol that allows measuring the activity of lysosomal hydrolases in digitonin-extracted cytosol and comparing it to total cellular activity to determine the extent of LMP.

1. The use of digitalis saponins (a detergent) to displace cholesterol to form pores in the cell membrane.
2. The difference in cholesterol content between the plasma membrane and the lysosomal membrane allows the concentration of digitalis saponin to penetrate the plasma membrane, but the lysosomal membrane is left intact.
3. The activity of lysosomal hydrolases is measured to determine the extent of LMP.

Characteristics of This Approach

• Cathepsin activity was normalized to lactate dehydrogenase (LDH).
• Cathepsin activity levels were normalized to cytosolic protein levels using a dedicated protein assay kit.
• Caspase activity can be measured in parallel in whole cell extracts.
• Optimization of digitonin extraction takes approximately 1 hour.
• The process of measuring LMP by digoxigenin extraction takes 2-2.5 hours.

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Reference

1. Jäättelä M, Nylandsted J. (2015) Quantification of Lysosomal Membrane Permeabilization by Cytosolic Cathepsin and β-N-Acetyl- Glucosaminidase Activity Measurements. Cold Spring Harb Protoc. 2015(11):1017-23.