THE PURIFICATION AND PARTIAL CHARACTERIZATION OF BONE RESORPTIVE POLYPEPTIDES FROM BOVINE BONE-MATRIX

Matrix proteins were extracted from bovine cortical bone with EDTA/Tri s-HCl under non-dissociative conditions at neutral pH. Four distinct b one resorptive proteins with molecular masses of 14, 25, 29 and 40 kDa were purified and partially characterized using an in vitro neonatal mouse calvarial assay and a growth factor assay using BALB/c/3T3 cells . The 14 kDa protein was purified by anion exchange chromatography (Mo no Q) and gel filtration (Superdex 75HR) using FPLC (fast protein liqu id chromatography); this factor stimulated the proliferation of MCF-7 human breast cancer cells, a bioassay which is specific for the insuli n-like growth factors (IGFs). The 25, 29 and 40 kDa proteins were puri fied by sequential chromatography as follows: anion-exchange (Mono Q), heparin-Sepharose, hydroxyapatite, concanavalin A-Sepharose, phenyl-S uperose, reversed phase high performance liquid chromatography (HPLC) and sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE) . The 25 kDa protein was identified as TGF-beta by its inhibitory effe ct on the proliferation of mink lung cells. The 40 kDa protein enhance d the formation of multinucleate tartrate-resistant acid phosphatase p ositive cells in a murine bone marrow differentiation assay, but was w ithout effect in an isolated osteoclast assay and had no growth factor activity; this protein is likely to be a colony stimulating factor. T he 29 kDa protein was also without growth factor activity; it was, how ever, able to stimulate bone resorption in the isolated osteoclast ass ay, suggesting a direct action in osteoclast function. The 29 and 40 k Da proteins may be osteoblast gene products that have been sequestrate d by the bone matrix in a similar fashion to TGF-beta and the IGFs. Th is is the first report of proteins isolated from bone matrix which dir ectly stimulate osteoclast differentiation and activity.