PURIFICATION AND CHARACTERIZATION OF CATHEPSIN-D FROM NORMAL HUMAN BREAST-TISSUE

The lysosomal aspartyl protease cathepsin D is present in most mammali an cells and is active in the catabolism of intracellular and endocyto sed proteins. It appears to be overexpressed and abnormally secreted i n breast cancer cells, and may contribute to the process of tumor meta stasis. In the present study, cathepsin D was purified 4500-fold from normal human breast tissue using pepstatin-agarose, DEAE Sephadex, and Sephadex G-75 chromatography. The resulting enzyme on SDS-PAGE contai ned five protein bands (47, 31, 29, 13, and 12 kDa) which were all imm unoreactive on western blot analysis using anti-cathepsin D polyclonal antibodies. The isoform profile of purified cathepsin D consisted of three major peaks at approximate pI 7.3, 6.8, and 6.3, and a broad are a of lower activity between pI of 5.0 and 2.0. The purified enzyme had a broad pH optimum centered around pH 3.3. Lectin blotting indicated that cathepsin D is a glycoprotein which is recognized by Galanthus ni valis agglutinin and concanavalin A, suggesting the presence of mannos e residues. However, Sambucus nigra agglutinin, Tetragonolobus purpure as agglutinin, Triticum vulgaris agglutinin, and Erythrina cristagalli agglutinin failed to recognize cathepsin D, suggesting a lack of lect in-available sialic acid, fucose, N-acetylglucosamine, and galactose r esidues, respectively.