Quantification of cathepsins B and L in cells.
A method for quantifying active cysteine proteinases in mammalian cells has been developed using an active-site-directed inhibitor. Fluoren-9-ylmethoxycarbonyl(di-iodotyrosylalanyl)-diaz omethane (Fmoc-[I2]Tyr-Ala-CHN2) was prepared and shown to react irreversibly with cathepsins B and L, but not with cathepsin S. The non- and mono-iodo forms of the inhibitor reacted with all three enzymes. These results demonstrate that, unlike cathepsins B and L, cathepsin S has a restricted S2-binding site that cannot accommodate the bulky di-iodotyrosine. Fmoc-[I2]Tyr-Ala-CHN2 was able to penetrate cells and react with active enzymes within the cells. A radiolabelled form of the inhibitor was synthesized and the concentration of functional inhibitor was established by titration with papain. This inhibitor was used to quantify active cysteine proteinases in cultured cells. Active cathepsin B was found to be expressed by all of the cells studied, consistently with a housekeeping role for this enzyme. Active forms of cathepsin L were also expressed by all of the cells, but in different quantities. Two additional proteins were labelled in some of the cells, and these may represent other non-characterized proteinases. Higher levels of active cathepsins B and L, and an unidentified protein of Mr 39000, were found in breast tumour cells that are invasive, compared with those that are not invasive. From the data obtained, it can be calculated that the concentrations of both active cathepsins B and L in lysosomes can be as high as 1 mM, each constituting up to 20% of total protein in the organelle. This new technique provides a more direct procedure for determining the proteolytic potential of cellular lysosomes.
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Division of Developmental Biology, Nemours Research Programs, Alfred I. duPont Hospital for Children, P.O. Box 269, Wilmington, DE 19899, USA.
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D001391:Azo Compounds / Q000494:pharmacology; D001665:Binding Sites; D001943:Breast Neoplasms / Q000201:enzymology; D002401:Cathepsin B / Q000032:analysis*; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis* / Q000378:metabolism; D002463:Cell Membrane Permeability; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes / Q000032:analysis; D008247:Lysosomes / Q000201:enzymology; D015394:Molecular Structure; D014407:Tumor Cells, Cultured
D001391:Azo Compounds; D004791:Enzyme Inhibitors; D007457:Iodine Radioisotopes; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C028217:cathepsin S