Isolation of a 25kDal, fibronectin-binding, mitogenic peptide from bovine plasma

In addition to fibronectin-derived peptides, several immunologically distinct peptides have been found to co-purify with fibronectin on gelatin-sepharose affinity chromatography of bovine plasma. We have isolated a 25kdal peptide ("25K") which co-purifies with fibronectin and which is mitogenic for cultured fibroblasts. 25K migrated as a doublet of Mr 25kdal and 25-28kdal on SDS-PAGE and stimulated 3H-thymidine incorporation into quiescent 3T3 fibroblasts. At 8 micrograms/ml, its mitogenic activity was found to be equivalent to 25 ng/ml PDGF, or 50% that of 10% foetal calf serum. It is unlikely that 25K is a proteolytic fragment of fibronectin since no cross-reactivity was found by western blotting and immunological staining, using a polyclonal anti-fibronectin antibody. Using affinity chromatography, it was found that 25K bound only weakly to gelatin, but inhibited the binding of fibronectin to gelatin. 25K bound strongly to fibronectin, an interaction inhibited by the presence of free gelatin. It would therefore appear that 25K binds specifically to fibronectin through the collagen-binding site.