Identification of lysyl oxidase and TRAMP as the major proteins in dissociative extracts of the demineralized collagen matrix of porcine dentine.

Carbonated apatite (dahllite) is formed within and between collagen fibrils in the mineralization of connective tissues. However, the mechanism of crystal nucleation at these sites has not been resolved. To identify non-collagenous proteins that may be involved in the nucleation process we have utilized a dissociative extraction procedure to isolate proteins associated non-covalently with the de-mineralized collagen matrix of dentine isolated from tooth roots of adult porcine incisors.

Biosynthesis of bone proteins by fetal porcine calvariae in vitro. Rapid association of sulfated sialoproteins (secreted phosphoprotein-1 and bone sialoprotein) and chondroitin sulfate proteoglycan (CS-PGIII) with bone mineral.

To study the biosynthesis of bone proteins, fragments of fetal porcine calvariae were cultured in the presence of 50 micrograms/ml ascorbate and 10 mM beta-glycerophosphate and individual cultures labeled for either 4 h or 48 h with [35S]-methionine, Na2[35SO4], Na3[32PO4] or [14C]-glycine plus [14C]-proline. The radiolabeled proteins in tissue extracts were obtained by sequential extraction with 4 M GuHCl (G1-extract), 0.5 M EDTA (E-extract), and again with 4 M GuHCl (G2-extract) and analyzed together with the radiolabeled proteins secreted into the medium.

Characterization of fetal porcine bone sialoproteins, secreted phosphoprotein I (SPPI, osteopontin), bone sialoprotein, and a 23-kDa glycoprotein. Demonstration that the 23-kDa glycoprotein is derived from the carboxyl terminus of SPPI.

Demineralizing extracts of porcine bone contain two large 66-80-kDa sialoproteins and smaller 20- and 23-kDa glycoproteins with similar chemical properties. Each protein was characterized following extraction from fetal calvariae and purification under dissociative conditions using Sepharose CL-6B, followed by fast protein liquid chromatography fractionation on hydroxyapatite and Mono Q resins. Unlike the large sialoproteins, the 20- and 23-kDa glycoproteins did not contain sialic acid.

Immunohistochemical localization of SPARC (osteonectin) and denatured collagen and their relationship to remodelling in rat dental tissues.

To study this relationship, specific antibodies were used to determine the distribution of these proteins in mature rat dental tissues. Staining for SPARC with affinity-purified polyclonal antibodies was prominent throughout molar and incisor ligaments, endosteal tissue, dental pulp and muscle. More moderate staining was observed in other soft tissues including the lamina propria of gingiva, whereas the staining of demineralized bone was weak and in dentine was barely detectable.

Characterization of multiple forms of small collagenous apatite-binding proteins in bone.

A number of small (Mrs 25-28 kDa) collagenous apatite-binding (SCAB) proteins that stain blue with 'Stains-All' have been isolated from fetal porcine bone by sequential extractions with 4M GuHCl (G1), followed by 0.5M EDTA (E), and again with 4M GuHCl (G2). Following purification under dissociative conditions, two types of SCAB proteins both with approximately one-third of their structure being collagenous, were identified in the EDTA extract.