Isolation of a nontransforming bone-derived growth factor from medium conditioned by fetal rat calvariae

Abstract

Previous studies have indicated that medium conditioned by 21-day-old fetal rat calvariae contains bioactive proteins termed bone-derived growth factors (BDGF) I and II. In the present studies we have purified the nontransforming BDGF II by dialysis, molecular sieving, three reverse phase HPLC steps, and preparative polyacrylamide gel electrophoresis. The second HPLC step (HPLC-2) yielded a recovery of 22% of the biological activity and achieved a 1500-fold purification, resulting in 20 micrograms protein/liter calvarial conditioned medium; the third HPLC step was of limited value in the purification of BDGF. Analytical PAGE revealed that the majority of the protein in HPLC-2-purified BDGF migrated with a relative molecular mass (Mr) of 11,000 and two additional proteins were seen at a Mr of 22,000-23,000. On preparative PAGE, the material migrating with a Mr of 11,000 stimulated parameters of bone and fibroblast growth in vitro, whereas the material with a Mr of 22,000-23,000 had less biological activity. Isoelectric focusing revealed that BDGF had an isoelectric point (pI) of 5. BDGF enhanced the incorporation of [3H]thymidine into DNA in fibroblast and calvarial cultures and of [3H]proline into collagen and noncollagen protein in calvariae. In conclusion, fetal rat calvariae secrete a BDGF with an estimated Mr of 11,000 and a pI of 5; this material stimulates bone and fibroblast growth in vitro.