The aspartyl protease cathepsin D (EC 3.4.23.5) appears to be found in incr
eased amounts and/or abnormally secreted in breast cancer cells, and may co
ntribute to the metastatic spread of malignancy. In the present study, cath
epsin D was purified 4800-fold in 20% yield from malignant human breast tis
sue using affinity chromatography on pepstatin-agarose and DEAE-Sephadex ch
romatography. Slab gel SDS-PAGE of the purified cathepsin D indicated the p
resence of three major protein bands (31, 13, 12, kDa) and two minor protei
n bands (47, 29 kDa). Western blotting indicated that the 31 kDa band was t
he major immunoreactive species. Isoelectric focusing indicated that the pu
rified cathepsin D consisted of three major isoforms at approximate pIs of
7.4, 7.0 and 6.6, and a possible isoform of lower activity centered around
pI 3.2. The pH curve of purified cathepsin D indicated a broad optimum cent
ered around pH 3.4. Lectin blotting suggested the presence of mannose resid
ues but no evidence was found for lectin-available sialic acid, fucose, N-a
cetylglucosamine and galactose residues. The investigated properties of pur
ified cathepsin D from malignant breast tissue are very similar, if not ide
ntical, to the properties of cathepsin D previously purified from normal hu
man breast tissue. Our findings suggest that the elevated activity and anti
genic levels of cathepsin D in malignant breast tissue are due to increased
amounts of apparently normal enzyme.