EDTA-INSOLUBLE, CALCIUM-BINDING PROTEOGLYCAN IN BOVINE BONE

A calcium ion precipitable, trypsin-generated proteoglycan fragment ha s been isolated from the demineralized, EDTA-insoluble matrices of bon e. The demineralized matrix was completely digested with trypsin, incr easing concentrations of CaCl2 were added to the supernatant, and the resulting precipitates were analyzed. The amount of precipitate gradua lly increased with higher concentrations of calcium and was reversibly solubilized by EDTA. After molecular sieve and anion exchange chromat ography, a proteoglycan-containing peak was obtained. Immunochemical a nalysis showed that this peak contained chondroitin 4-sulfate and poss ibly keratan sulfate. Amino acid analysis showed that this proteoglyca n contained high amounts of aspartic acid/asparagine (Asx), serine (Se r), glutamic acid/glutamine (Glx), proline (Pro), and glycine (Gly); h owever, it contained little leucine (Leu) which suggests that it is no t a member of the leucine-rich small proteoglycan family. In addition, significant amounts of phosphoserine (P-Ser) and hydroxyproline (Hyp) were identified in hydrolysates of this fraction. A single band (Mr 5 9 kDa) was obtained on SDS-PAGE that stained with Stains-all but not w ith Coomassie Brilliant Blue R-250. If bone powder was trypsinized pri or to demineralization, this proteoglycan-containing fraction was not liberated. Collectively, these results indicate that a proteoglycan oc curs in the demineralized matrix that is precipitated with CaCl2 and i s closely associated with both mineral and collagen matrices. Such a m olecule might facilitate the structural network for the induction of m ineralization in bone.