| Abstract: | Bone sialoprotein (BSP) was shown to be a potent nucleator of hydroxy apatite (HA) in a steady-state agarose gel system (Hunter and Goldberg, 1993, PNAS 90: 8562). Nucleation of HA was also demonstrated with the homopolymer poly-glutamic acid but not with poly-aspartic acid or osteopontin. Since BSP contains contiguous sequences of glutamic acid, it is reasonable to suggest that the HA-nucleating activity of BSP resides within these regions. Purified porcine BSP was treated with trypsin and digests fractionated by gel filtration. In addition to small peptides (P3–5), two peptides of 38 kDa (PI) and 25 kDA (P2) were recovered, and after characterization assigned to the regions within BSP encompassing residues 133–272 (PI) and 42–125 (P2). Each of these peptides contained one of the two glutamic acid-rich regions of porcine BSP. In the steady-state agarose gel system, BSP, PI and P2 induced HA formation, whereas the pooled small BSP-derived peptides (P3–5) did not. Analysis by circular dichroism spectroscopy revealed that the homopolymer poly-L-glutamic acid assumes a helical structure, while poly-L-aspartic acid does not. These findings suggest that the nucleating activity does not require intact molecules, that the nucleation of HA by BSP appears to require glutamic acid-rich sequences in a helical conformation and that there are two domains in porcine BSP that are each capable of nucleating HA. |
