title: Western blotting and isoform analysis of cathepsin D from normal and malignant human breast cell lines.
abstract: 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.
authors.affiliation: Department of Chemistry, MTC, Lehigh University, Bethlehem, PA 18015, USA.
authors.forename: L D
authors.initials: LD
authors.lastname: Laury-Kleintop
authors.forename: E C
authors.initials: EC
authors.lastname: Coronel
authors.forename: M K
authors.initials: MK
authors.lastname: Lange
authors.forename: T
authors.initials: T
authors.lastname: Tachovsky
authors.forename: S
authors.initials: S
authors.lastname: Longo
authors.forename: S
authors.initials: S
authors.lastname: Tucker
authors.forename: J A
authors.initials: JA
authors.lastname: Alhadeff
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000737:chemistry* / Q000201:enzymology; D002402:Cathepsin D / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007536:Isomerism; D007700:Kinetics; D010436:Pepstatins / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D004792:Enzyme Precursors; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D; C031375:pepstatin