[Hydroxyapatite-binding phosphoproteins synthesized by osteoblasts and fibroblasts].

The present study attempted to explore the newly synthesized phosphoproteins secreted by osteoblastlike cells and fibroblasts from rat pup calvaria and skin. The phosphoproteins, which are non-collagenous proteins, are posttranslationally phosphorylated by the kinases and secreted into the extracellular matrix and medium in the osteoblast-like cell and fibroblast cultures. The osteoblast-enriched cell population and fibroblast isolated from 2 day-old rat calvaria and skin by sequential enzymatic digestion were cultured in a defined medium containing fetal calf serum protein ( 2mg/ml) . At confluence, these cells and fibroblasts obtained from the 3rd isolated cells were incubated with 32Pi (Na2H32PO4, NEX-011) for 24 hr. The TCA precipitates of the medium were applied to hydroxyapatite column eluted by a stepwise of 6 mM, 0.1 M, 0.5M and 1.5M Pi Hepes buffer pH 7.4 containing 3M urea and 5mM levamisole. The 0.5M Pi eluted fractions of osteoblast-like cells and fibroblasts were electrophoresed on a 10% SDS-polyacrylamide gels for autoradiogram. The molecular weight of hydroxyapatite binding phosphoproteins synthesized by cultured cells were estimated 75K, 66K, 58K, 38K and 15.5K for osteoblast-like cells and 15.5 K for fibroblasts. The phosphoproteins, which can bind tightly on hydroxyapatite, may regulate the mineralizing processes.