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Quantification of cathepsins B and L in cells
R Xing1, A K Addington, R W Mason
1Division of Developmental Biology, Nemours Research Programs, Alfred I. duPont Hospital for Children, P.O. Box 269, Wilmington, DE 19899, USA.
The Biochemical Journal
|June 26, 1998
Summary
A new method quantifies active cysteine proteinases in cells using a specific inhibitor. This technique reveals varying levels of cathepsins B and L, with higher amounts in invasive breast tumor cells.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Cysteine proteinases are crucial enzymes in mammalian cells.
- Quantifying active forms of these enzymes is essential for understanding cellular processes.
- Existing methods may lack specificity or cellular penetration.
Purpose of the Study:
- To develop and validate a novel method for quantifying active cysteine proteinases within mammalian cells.
- To characterize the substrate specificity of a newly synthesized active-site-directed inhibitor.
- To assess the levels of active cathepsins B and L in various cell types, including comparison of invasive and non-invasive breast tumor cells.
Main Methods:
- Synthesis and characterization of Fluoren-9-ylmethoxycarbonyl(di-iodotyrosylalanyl)-diaz omethane (Fmoc-[I2]Tyr-Ala-CHN2), an active-site-directed inhibitor.
- Testing the inhibitor's reactivity with cathepsins B, L, and S, and its ability to penetrate cells.
- Utilizing a radiolabeled form of the inhibitor for quantification of active cysteine proteinases in cultured cells.
- Titration with papain to establish the concentration of functional inhibitor.
Main Results:
- Fmoc-[I2]Tyr-Ala-CHN2 selectively inhibited cathepsins B and L, but not cathepsin S, due to cathepsin S's restricted S2-binding site.
- The inhibitor successfully penetrated cells and reacted with intracellular active enzymes.
- Active cathepsin B was ubiquitously expressed, suggesting a housekeeping role.
- Variable expression levels of active cathepsin L were observed across cell types.
- Invasive breast tumor cells showed higher levels of active cathepsins B and L and an unidentified 39 kDa protein compared to non-invasive cells.
- Lysosomal concentrations of active cathepsins B and L can reach up to 1 mM, comprising 20% of organelle protein.
Conclusions:
- A novel, cell-penetrating inhibitor allows for direct quantification of active cysteine proteinases.
- The findings highlight the ubiquitous role of cathepsin B and differential roles of cathepsin L in cellular function.
- Elevated levels of specific cysteine proteinases correlate with breast tumor invasiveness.
- This method provides a direct assessment of the proteolytic potential within cellular lysosomes.