An analysis of the biochemical and biosynthetic properties of dentin phosphoprotein.
Dentin phosphoprotein (DPP) and dephosphorylated DPP (deP-DPP) were isolated from developing bovine incisor and purified by DEAE-Sephacel and Sepharose CL-6B chromatography. Intact DPP showed partial resistance to proteolytic cleavage; while deP-DPP was completely susceptible to protease digestion. This result suggested that phosphate residues within DPP molecule may protect its structural integrity. DPP was also very sensitive to heat. When DPP was heated at 100 degrees C in the presence of sodium dodecyl sulfate, increasing concentrations of SDS resulted in a protective effect from degradation. An analysis of DPP biosynthesis was carried out by isolation of the odontoblast tissue/cells attached to forming dentin or scraped from dentin surface followed by incubation with radioactive amino acids and phosphate. DPPs produced in vitro were larger in size than those DPPs extracted from mature dentin matrix. To determine the size of the unprocessed form of DPP, Xenopus laevis oocytes were incubated in [32P]-phosphate-containing medium after microinjection of poly(A)+ mRNA extracted from the bovine odontoblasts. Radioactive DPP analyzed by two-dimensional polyacrylamide gel electrophoresis and immunoprecipitation with an anti-DPP monoclonal antibody was shown to have an apparent Mr of 150-160 k. These data imply that changes in molecular size of DPP may occur by post-translational cleavage or other modification such as degradation within dentin matrix in vivo.
Ibaraki
K
K
Department of Biochemistry, School of Dentistry, Tokyo, Japan.
Shimokawa
H
H
Sasaki
S
S
D000469:Alkaline Phosphatase; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D002417:Cattle; D003804:Dentin / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D015151:Immunoblotting; D066298:In Vitro Techniques; D010710:Phosphates / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D011061:Poly A / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011868:Radioisotopes; D014982:Xenopus laevis
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D010710:Phosphates; D010750:Phosphoproteins; D012333:RNA, Messenger; D011868:Radioisotopes; C017630:phosphophoryn; D011061:Poly A; D000469:Alkaline Phosphatase; D010450:Endopeptidases