The purification and partial characterization of bone resorptive polypeptides from bovine bone matrix.
Matrix proteins were extracted from bovine cortical bone with EDTA/Tris-HCl under non-dissociative conditions at neutral pH. Four distinct bone 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 (Mono Q) and gel filtration (Superdex 75HR) using FPLC (fast protein liquid chromatography); this factor stimulated the proliferation of MCF-7 human breast cancer cells, a bioassay which is specific for the insulin-like growth factors (IGFs). The 25, 29 and 40 kDa proteins were purified by sequential chromatography as follows: anion-exchange (Mono Q), heparin-Sepharose, hydroxyapatite, concanavalin A-Sepharose, phenyl-Superose, 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 effect on the proliferation of mink lung cells. The 40 kDa protein enhanced the formation of multinucleate tartrate-resistant acid phosphatase positive cells in a murine bone marrow differentiation assay, but was without effect in an isolated osteoclast assay and had no growth factor activity; this protein is likely to be a colony stimulating factor. The 29 kDa protein was also without growth factor activity; it was, however, able to stimulate bone resorption in the isolated osteoclast assay, suggesting a direct action in osteoclast function. The 29 and 40 kDa proteins may be osteoblast gene products that have been sequestrated by the bone matrix in a similar fashion to TGF-beta and the IGFs. This is the first report of proteins isolated from bone matrix which directly stimulate osteoclast differentiation and activity.
Hill
P A
PA
Cell and Molecular Biology Department, Strangeways Research Laboratory, Worts Causeway, Cambridge, UK.
Reynolds
J J
JJ
Meikle
M C
MC
D000818:Animals; D001853:Bone Marrow / Q000187:drug effects; D001857:Bone Matrix / Q000737:chemistry*; D001862:Bone Resorption; D002417:Cattle; D002454:Cell Differentiation; D004492:Edetic Acid; D006133:Growth Substances / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D051379:Mice; D010010:Osteoclasts / Q000187:drug effects; D011506:Proteins / Q000302:isolation & purification*
D006133:Growth Substances; D011506:Proteins; D004492:Edetic Acid