Objectives: Recently we used a steady-state gel system which allowed the diffusion of Ca and PO₄ ions into agarose gels which contained different molecules. This allowed us to examine the molecules' ability to nucleate mineral. We reported that phosphophoryn's repeated motifs containing high percentage of serine were capable of precipitating more Ca & PO₄ than the polypeptide made from the N-terminal sequences of phosphophoryn. What was not observed was whether this Ca & PO₄ precipitate was hydroxyapatite. We therefore repeated this experiment using the same apparatus and two synthetic polypeptides made from human phosphophoryn's sequences: DDPNSSDESNGNDD (from N terminal of phosphophoryn, P1) and DSKSDSSKSESDSS (from repeated sequence of the serine rich motif of phosphophoryn, P2). Methods: Two reservoirs, one containing 10 mM CaCl₂ and the other containing 10 mM Na₂HPO₄, were connected to a series of horizontal glass tubes. P1 or P2 or phosphorylated P2 (by casein kinase 1 and 2) was mixed with 1% of agarose gel at a concentration of 10 ug /ml and added to the glass tubes. The system allowed Ca and PO₄ ions to diffuse independently into the opposite sides of the gels. After 6 days of incubation at 37^oC, the gels were analyzed using x-ray diffraction. Results: The gel containing P1 or P2 molecule showed no evidence of hydroxyapatite formation. However, the x-ray diffraction analysis identified the formation of hydroxyapatite in the phosphorylated P2 containing gel. Conclusion: Thus we conclude that phosphophoryn's ability to nucleate hydroxyapatite is dependent on its repeated phosphorylated serine containing motifs. Supported by NIH grants DE 12899

Back to the Mineralized Tissue Program
Back to the IADR/AADR/CADR 82nd General Session (March 10-13, 2004)

Top Level Search