Binding of bone sialoprotein, osteopontin and synthetic polypeptides to hydroxyapatite.

The phosphorylated acidic glycoproteins bone sialoprotein (BSP) and osteopontin (OPN) bind to hydroxyapatite (HA) crystals and may be involved in the regulation of bone mineralization. The HA-binding properties of these proteins have been attributed to glutamic acid-rich sequences in BSP and aspartic acid-rich sequences in OPN. The present study examines the roles of these polycarboxylate sequences in the binding of BSP and OPN to HA. Porcine BSP, OPN and the synthetic polypeptides poly-L-glutamic acid [Poly(Glu)] and poly-L-aspartic acid [Poly(Asp)] were labeled with fluorescein isothiocyanate and their binding to HA determined by fluorimetry. From the binding isotherms, dissociation constants (KDs) for all the reagents tested were determined to be in the micromolar range. The saturation binding capacities of HA for Poly(Glu), Poly(Asp), BSP and OPN were similar (500-600 micrograms/m2). To investigate the role of glutamic acid-rich and aspartic acid-rich sequences in the binding to HA of BSP and OPN, respectively, competitive binding studies with Poly(Glu) and Poly(Asp) were performed. Poly(Glu) was able to displace a maximum of 100% of Poly(Glu), 81% of OPN, 68% of BSP and 65% of Poly(Asp). Poly(Asp) was able to displace a maximum of 100% of Poly(Glu), 99% of Poly(Asp), 95% of OPN and 89% of BSP. These results are consistent with the view that BSP and OPN bind to HA via their polycarboxylate sequences, but suggest a complex mode of interaction between polyelectrolytes and ionic crystals.

The staining of acidic proteins on polyacrylamide gels: enhanced sensitivity and stability of "Stains-all" staining in combination with silver nitrate.

A number of acidic proteins, such as those found in bone and dentin, are poorly resolved on acrylamide gels using Coomassie blue or silver nitrate staining. The cationic dye Stains-all allows visualization and identification of these proteins due to their differential staining: highly acidic proteins stain blue and intact proteoglycans stain purple, whereas less acidic proteins stain pink. However, the use of Stains-all is limited due to relatively poor staining sensitivity and lack of stability to light. A procedure which addresses these deficiencies has been developed utilizing established protocols for Stains-all staining followed by silver nitrate incubation and development. In this way, phosphoproteins such as osteopontin, bone sialoprotein, dentin phosphophoryn, and other acidic glycoproteins are visualized at higher sensitivity (greater than fivefold) and staining stability than normally achieved with just Stains-all. The protocol stains a greater variety of proteins than a combined alcian blue/silver staining procedure previously described. Utilizing the Stains-all/silver protocol, porcine bone osteopontin, a protein not visualized by standard silver staining, can be observed in amounts as little as 0.25 ng on polyacrylamide gels. Furthermore, densitometric scans demonstrate that the staining intensity is proportional to osteopontin amount and can be used for quantification over a range from 0.25 to 50 ng.

Determination of the hydroxyapatite-nucleating region of bone sialoprotein.

Bone sialoprotein (BSP) was shown to be a potent nucleator of hydroxyapatite (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 (P1) and 25 kDA (P2) were recovered, and after characterization assigned to the regions within BSP encompassing residues 133-272 (P1) 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, P1 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 and 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.

Cardiac amyloidoma with IgM-kappa gammopathy.

A 73-year-old man presented with dyspnea and atrial flutter associated with an amyloid tumor in the heart. IgM-kappa gammopathy, hypercalcemia, and extensive cardiac and mediastinal invasion suggested a malignant lymphoid or plasma cell process. Although amyloidoma is generally considered to be a benign tumor, the aggressive features of this case mandated chemotherapy because the critical location rendered the tumor inoperable. This case provides noteworthy evidence in support of a possible pathogenic relationship between amyloidoma and plasmacytoma by virtue of dual representative features: localized amyloid infiltrated with plasma cells and the associated gammopathy. Local and systemic malignant features lend additional support to this hypothesis.