Biochemical stability of a bone-inducing substance from murine osteosarcoma.

Devitalized cultured cells of a murine osteosarcoma preserved strong osteogenic activity even after incubation in neutral and acidic buffer at 37 degrees for six days. This observation suggested that an osteosarcoma-derived osteogenic factor was considerably stable to endogenous proteases, or that osteosarcomas do not synthesize enzymes for degradation of the osteogenic factor. EDTA decalcification or extraction of the osteosarcoma at 37 degrees for six days had no apparent effect on the osteogenic factor. Disulfide-bond reducing agents (2-mercaptoethanol and dithiothreitol) completely inactivated the osteogenic activity and in the presence of guanidine HCl, the loss of activity was irreversible by reoxidation. There findings support the view that disulfide-bonds in the molecular structure of the osteogenic factor are essential for the biological activity.