The mitogenic activity of a heparin-binding fibronectin fragment (Mr 35,000) produced by cathepsin D digestion

Fibronectin was partially digested using the lysosomal proteinase cathepsin D and the fragments produced tested for their effect on the rate of DNA synthesis in quiescent Chinese Hamster Fibroblasts (CH23). A mitogenically active digest was produced after digestion of fibronectin with cathepsin D for 2 to 3h at a 1:200 (w/w) ratio of enzyme to substrate at 37 degrees C. Affinity chromatography of the active digest on both gelatin and heparin 4BCl columns demonstrated that the active fragment was gelatin-nonbinding but heparin-binding, and therefore derived from one of the two or three heparin binding domains of intact fibronectin. The active fragment was found to have a Mr35,000 by gel filtration on sephacryl S200. A dose-response curve of the mitogenic fragment purified by gel filtration against DNA synthesis showed it to be maximally active at a concentration of approximately 10 micrograms/ml.