QUANTIFICATION OF CATHEPSIN-B AND CATHEPSIN-L IN CELLS

A method for quantifying active cysteine proteinases in mammalian cell s has been developed using an active-site-directed inhibitor. methoxyc arbonyl(di-iodotyrosylalanyl)-diazomethane (Fmoc-[I-2]Tyr-Ala-CHN2) wa s prepared and shown to react irreversibly with cathepsins B and L, bu t 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 S-2-binding site tha t cannot accommodate the bulky di-iodotyrosine. Fmoc-[I-2]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 c oncentration of functional inhibitor was established by titration with papain. This inhibitor was used to quantify active cysteine proteinas es in cultured cells. Active cathepsin B was found to be expressed by all of the cells studied, consistently with a housekeeping role for th is enzyme. Active forms of cathepsin L were also expressed by all of t he cells, but in different quantities. Two additional proteins were la belled in some of the cells, and these may represent other noncharacte rized proteinases. Higher levels of active cathepsins B and L, and an unidentified protein of M-r 39 000, 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 a ctive cathepsins B and L in lysosomes can be as high as 1 mM, each con stituting up to 20% of total protein in the organelle. This new techni que provides a more direct procedure for determining the proteolytic p otential of cellular lysosomes.