Advertisement

Calcified Tissue International

, Volume 33, Issue 1, pp 385–394 | Cite as

Purification, composition, and31P NMR spectroscopic properties of a noncollagenous phosphoprotein isolated from chicken bone matrix

  • Sandra L. Lee
  • Melvin J. Glimcher
Article

Summary

Fractionation of the EDTA-soluble, noncollagenous proteins of the organic matrix of chicken bone by Sephadex G-100 molecular sieving has revealed that the majority of the organic phosphorus is present in two fractions, from one of which a homogeneous phosphoprotein has been isolated. The purified phosphoprotein has an apparent molecular weight of 12,000 and contains bothO-phosphoserine andO-phosphothreonine.31P-NMR spectroscopy demonstrates that all of the organic phosphorus exists in the form of phosphomonoesters which have an average pK2 of 6.8. The phosphoprotein is highly acidic due to its high content of dicarboxylic acids in addition to the presence of organic phosphorus. The characteristic amino acid composition of the phosphoprotein establishes its noncollagenous nature and highlights the differences among bone, dentin, and enamel phosphoproteins. The absence ofγ-carboxyglutamic acid distinguishes it from osteocalcin, the noncollagenousγ-carboxyglutamic acid-containing peptide of bone matrix.

Key words

Phosphoprotein Bone 31P-NMR Phosphoserine Phosphothreonine 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Glimcher, M. J.: Phosphopeptides of enamel matrix, J. Dent. Res.58B: 790–806, 1979Google Scholar
  2. 2.
    Glimcher, M. J.: Composition, structure and organization of bone and other mineralized tissues and the mechanism of calcification. In R. O. Greep, E. B. Astwood (eds.): Handbook of Physiology: Endocrinology, Vol. 7, pp. 25–116. American Physiological Society, Washington, D.C., 1976Google Scholar
  3. 3.
    Veis, A.: The role of acidic proteins in biological mineralizations. In D. H. Everett, B. Vincent (eds.): Ions in Macromolecular and Biological Systems, Colston Paper No. 29, pp. 259–272. Scientechnia, Bristol, 1978Google Scholar
  4. 4.
    Cohen-Solal, L., Lian, J. B., Kossiva, D., Glimcher, M. J.: The identification ofO-phosphothreonine in the soluble non-collagenous phosphoproteins of bone matrix, FEBS Lett.89:107–110, 1978CrossRefPubMedGoogle Scholar
  5. 5.
    Cohen-Solal, L., Lian, J. B., Kossiva, D., Glimcher, M. J.: Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken bone matrix: the presence ofO-phosphoserine andO-phosphothreonine in non-collagenous proteins, and their absence from phosphorylated collagen, Biochem. J.177:81–98, 1979PubMedGoogle Scholar
  6. 6.
    Lee, S. L.: Calcium ion binding and conformational properties of bovine dentin phosphoprotein and related synthetic polyamino acids. Doctoral thesis, Northwestern University, Evanston, Ill., 1977Google Scholar
  7. 7.
    Lee, S. L., Veis, A., Glonek, T.: Dentin phosphoprotein, Biochemistry16:2971–2977, 1977CrossRefPubMedGoogle Scholar
  8. 8.
    Veis, A., Spector, A. R., Zamoscianyk, H.: The isolation of an EDTA-soluble phosphoprotein from mineralizing bovine dentin, Biochim. Biophys. Acta257:404–413, 1972PubMedGoogle Scholar
  9. 9.
    Spector, A. R.: A phosphorus-containing protein from an actively mineralizing tissue. Doctoral thesis, Northwestern University, Evanston, Ill., 1969Google Scholar
  10. 10.
    Spector, A. R., Glimcher, M. J.: The extraction and characterization of soluble anionic phosphoproteins from bone, Biochim. Biophys. Acta263:593–603, 1972Google Scholar
  11. 11.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the Folin phenol reagent, J. Biol. Chem.193:265–275, 1951PubMedGoogle Scholar
  12. 12.
    Dische, Z.: Color reactions of nucleic acid components. In E. Chargaff, J. N. Davidson (eds.): The Nucleic Acids: Chemistry and Biology, Vol. I, pp. 285–305. Academic Press, New York, 1955Google Scholar
  13. 13.
    Brown, A. H.: Determination of pentose in the presence of large quantities of glucose, Arch. Biochem.11:269–278, 1946Google Scholar
  14. 14.
    Aminoff, D.: Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids, Biochem. J.81:384–392, 1962Google Scholar
  15. 15.
    Bitter, T., Muir, H. M.: A modified uronic acid carbazole reaction, Anal. Biochem.4:330–334, 1962CrossRefPubMedGoogle Scholar
  16. 16.
    Monson, J. M., Bornstein, P.: Identification of a disulfide-linked procollagen as the biosynthetic precursor of chickbone collagen, Proc. Natl. Acad. Sci. U.S.A.70:3521–3525, 1973PubMedGoogle Scholar
  17. 17.
    Ackers, G. K.: Molecular exclusion and restricted diffusion processes in molecular-sieve chromatography, Biochemistry3:723–730, 1964CrossRefPubMedGoogle Scholar
  18. 18.
    Bylund, D. B., Huang, T.-S.: Decomposition of phosphoserine and phosphothreonine during acid hydrolysis, Anal. Biochem.73:477–485, 1976CrossRefPubMedGoogle Scholar
  19. 19.
    Hauschka, P. V.: Quantitative determination of γ-carboxyglutamic acid in proteins, Anal. Biochem.80:212–223, 1977CrossRefPubMedGoogle Scholar
  20. 20.
    Laemmli, U. K., Favre, M.: Maturation of the head of bacteriophage T4. I. DNA packaging events, J. Mol. Biol.80:575–599, 1973CrossRefPubMedGoogle Scholar
  21. 21.
    Scott, P. G., Telser, A. G., Veis, A.: Semi-quantitative determination of cyanogen bromide peptides of collagen in SDS-polyacrylamide gels, Anal. Biochem.70:251–257, 1976CrossRefPubMedGoogle Scholar
  22. 22.
    Radin, N. S.: Preparation of lipid extracts, Methods Enzymol.14:245–254, 1969CrossRefGoogle Scholar
  23. 23.
    Roufosse, A., Strawich, E., Fossel, E., Lee, S., Glimcher, M. J.:31P nuclear magnetic resonance studies of E4 phosphopeptide of embryonic bovine enamel in solution, J. Dent. Res.58B:1019–1020, 1979Google Scholar
  24. 24.
    Seyer, J. M., Glimcher, M. J.: Isolation, characterization and partial amino acid sequence of a phosphorylated polypeptide (E4) from bovine embryonic dental enamel, Biochim. Biophys. Acta493:441–451, 1977PubMedGoogle Scholar
  25. 25.
    Cohen-Solal, L., Cohen-Solal, M., Glimcher, M. J.: Identification of γ-glutamyl phosphate in the α2 chains of chicken bone collagen, Proc. Natl. Acad. Sci. U.S.A.76:4327–4330, 1979PubMedGoogle Scholar
  26. 26.
    Papas, A., Seyer, J. M., Glimcher, M. J.: Isolation from embryonic bovine dental enamel of a polypeptide (E3) containing as its only phosphorylated sequence Glu-O-Phosphoserine-Leu, FEBS Lett.79:276–280, 1977CrossRefPubMedGoogle Scholar
  27. 27.
    Dimuzio, M. T., Veis, A.: Phosphophoryns: major non-collagenous proteins of rat incisor dentin, Calcif. Tissue Res.25:169–178, 1978CrossRefPubMedGoogle Scholar
  28. 28.
    Levine, P. T., Glimcher, M. J., Krane, S. M.: The identification and isolation of serine phosphate in the developing proteins of rodent enamel, Arch. Oral Biol.12:311–313, 1967CrossRefPubMedGoogle Scholar
  29. 29.
    Levine, P. T., Seyer, J., Huddleston, J., Glimcher, M. J.: The comparative biochemistry of the organic matrix proteins of developing enamel. I. Amino acid composition, Arch. Oral Biol.12:407–410, 1967CrossRefPubMedGoogle Scholar
  30. 30.
    Butler, W. T.: Dentinal phosphoproteins. In H. C. Slavkin (ed.): The Comparative Molecular Biology of Extracellular Matrices, pp. 255–262. Academic Press, New York, 1972Google Scholar
  31. 31.
    Butler, W. T., Finch, J. E., DeSteno, C. V.: Chemical character of proteins in rat incisors, Biochim. Biophys. Acta257:167–171, 1972PubMedGoogle Scholar
  32. 32.
    Glimcher, M. J., Kossiva, D., Roufosse, A.: Identification of phosphopeptides and γ-carboxyglutamic acid-containing peptides in epiphyseal growth plate cartilage, Calcif. Tissue Int.27:187–191, 1979PubMedGoogle Scholar
  33. 33.
    Glimcher, M. J., Lefteriou, B., Kossiva, D.: Identification ofO-phosphoserine,O-phosphothreonine and γ-carboxyglutamic acid in the non-collagenous proteins of bovine cementum; comparison with dentin, enamel and bone. Calcif. Tissue Int.28:83–86, 1979PubMedGoogle Scholar
  34. 34.
    Glimcher, M. J., Brickley-Parsons, D., Kossiva, D.: Phosphopeptides and γ-carboxyglutamic acid-containing peptides in calcified turkey tendon: their absence in uncalcified tendon. Calcif. Tissue Int.27:281–284, 1979PubMedGoogle Scholar
  35. 35.
    Dimuzio, M. T., Veis, A.: The biosynthesis of phosphophoryns and dentin collagen in the continuously erupting rat incisor, J. Biol. Chem.253:6845–6852, 1978PubMedGoogle Scholar
  36. 36.
    Lee, S. L., Glimcher, M. J.: Bone matrix phosphoproteins from adult avian metatarsals, J. Cell Biol.83:464a, 1979Google Scholar

Copyright information

© Springer-Verlag 1981

Authors and Affiliations

  • Sandra L. Lee
    • 1
  • Melvin J. Glimcher
    • 1
  1. 1.Department of Orthopaedic Surgery, Harvard Medical SchoolChildren's Hospital Medical CenterBostonUSA

Personalised recommendations