Cathepsin D from normal (Hs578Bst) and malignant (MCF7, MDA-MB-231) breast cell lines has been characterized with regard to its kinetic properties, activity levels, precursor and processed M(r) forms, and
isoform composition. Normal cell
cathepsin D appears to have a more neutral pH optimum (pH 3.5) than the
cancer cell line (pH 3.0-3.2) and greater activity between pH values of 4.0 to 4.5. The two
cancer cell lines have approximately 1.5 to 2.0-fold increased total
acid protease activity and 2 to 3-fold increased
pepstatin-inhibitable
protease activity (i.e.
cathepsin D) when compared to the normal breast cell line. Western blotting indicates that a major processed form of
cathepsin D for all three cell lines occurs at 31 kDa. The
cancer cell lines contain significant amounts of
cathepsin D precursors of 47 and 42 kDa whereas the normal cell line contains little if any of these precursors. Isoelectric focusing indicates that the normal cell line contains approximately 50% of its total
acid protease activity at pIs above 4 whereas the
cancer cell lines contain 70-80% of their
protease activity at such pIs. In addition, the
cancer cell lines contain two to three major
isoforms between pIs of 5.5 and 6.3 which were not present in the normal cell line. The
isoforms from pI values of 5.5 to 7.3 for all three cell lines are 100%
pepstatin-inhibitable. In addition, Western blot analysis indicates that these
isoforms contain the processed 31 kDa form of
cathepsin D. The combined results indicate that the two
breast cancer cell lines are similar to biopsied malignant breast tissue in exhibiting altered
acid protease isoform profiles with increased relative amounts of
pepstatin-inhibitable and immunoreactive
acid protease activity (
cathepsin D) compared to normal breast tissue or cells.