Purification and characterization of cathepsin D from normal human breast tissue.
The lysosomal aspartyl protease cathepsin D is present in most mammalian cells and is active in the catabolism of intracellular and endocytosed proteins. It appears to be overexpressed and abnormally secreted in breast cancer cells, and may contribute to the process of tumor metastasis. 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 contained five protein bands (47, 31, 29, 13, and 12kDa) which were all immunoreactive 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 area 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 nivalis agglutinin and concanavalin A, suggesting the presence of mannose residues. However, Sambucus nigra agglutinin, Tetragonolobus purpureas agglutinin, Triticum vulgaris agglutinin, and Erythrina cristagalli agglutinin failed to recognize cathepsin D, suggesting a lack of lectin-available sialic acid, fucose, N-acetylglucosamine, and galactose residues, respectively.
Wright
L M
LM
Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015, USA.
Levy
E S
ES
Patel
N P
NP
Alhadeff
J A
JA
D015153:Blotting, Western; D001940:Breast / Q000201:enzymology*; D002402:Cathepsin D / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D027801:Galanthus; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D037102:Lectins; D008970:Molecular Weight; D037121:Plant Lectins; D011480:Protease Inhibitors / Q000494:pharmacology
D006023:Glycoproteins; D037102:Lectins; D037121:Plant Lectins; D011480:Protease Inhibitors; D002402:Cathepsin D