Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human-breast cancer cells

Abstract

An estrogen‐regulated 52‐kDa glycoprotein secreted by MCF₇ breast cancer cells was first purified from serum‐free conditioned medium by concanavalin‐A–Sepharose (ConA–Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52‐kDa protein [Garcia et al. (1985) Cancer Res. 45, 709–716]. Using ConA–Sepharose and monoclonal antibody affinity chromatographies, the secreted 52‐kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‐PAGE) and one single N‐terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two‐step procedure, applied to MCF₇ cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250‐fold with a yield of 30%. These components were further separated by high‐performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver‐stained SDS‐PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52‐kDa protein and the cellular 34‐kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34‐kDa protein is given. Western immunoblotting, pulse chase studies and post‐translational studies indicate that the 52‐kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.