Identification of a 15,000-molecular-weight form of immunoreactive transforming growth factor alpha in extracts of porcine pituitary

Abstract

Two different mitogenic activities were identified from extracts of porcine pituitary by using COMMA-D mouse mammary epithelial cells in a serum-free 3H-thymidine incorporation assay. Porcine pituitaries were extracted in phosphate-buffered saline (pH 7.4) and 25-80% (NH4)2SO4 pellets were dialyzed and chromatographed by using DEAE-Sepharose chromatography (pH 8.0), resulting in two peaks (I and II) of mitogenic activity. Peak I represented a recovery of 73% of the units of mitogenic activity present in crude extract of pituitary while only 1.25% of the activity was recovered in peak II. Peak I was further purified by using CM-Sephadex and heparin-Sepharose chromatographies and yielded a mitogen that was able to elicit one-half-maximal stimulation of 3H-thymidine incorporation by COMMA-D cells at 48 pg/ml. As expected with pituitary as the tissue source, peak I was confirmed to be basic fibroblast growth factor (bFGF) by using specific antibodies in enzyme-linked immunosorbent assay and Western immunoblotting procedures. Peak II was further purified by using chromatofocusing (pH 7.3-5.0), reverse-phase, and cation-exchange HPLCs. The mitogenic activity eluted at pH 6.3 from chromatofocusing, migrated as a 13-kDa molecule on gel filtration HPLC, and did not bind to heparin-Sepharose under conditions which bound fibroblast growth factors. The material purified from peak II and rat synthetic transforming growth factor alpha (TGF alpha) competed in a parallel fashion with 125I-epidermal growth factor for receptor binding with A431 human epidermal carcinoma cells. In addition, the mitogen purified from peak II showed a single immunoreactive band migrating at 15 kDa when specific antiserum against TGF alpha was used in a Western immunoblotting procedure. The data suggest that in addition to the well-documented presence of bFGF, normal adult porcine pituitaries contain a 15-kDa form of immunoreactive TGF alpha that binds to EGF receptors and is mitogenic for mammary epithelial cells.