Abstruct
The originally mineralized area of a fixed and demineralized specimen of newborn rat calvarium was stained blue with Stains-all, which stained phosphoprotiens, glycoproteins and Ca-binding proteins blue. The material stained blue in the histological sections was solubilized by an acidic solution and found to be the protein (Stains-all positive protein = SAPP). This SAPP was found to consist of two different types of phosphoproteins, which had the same amino acid sequence at the N-terminal region and a quite similar amino acid composition to each other. The minor one was equal to the 44kD phosphoprotein which has previously been reported by Prince et al (J. Biol. Chem., 262, 2900, 1987). The major one was a 66kD phosphoprotein, it's molecular weight being determined by SDS electrophoresis on a 15% polyacrylamide gel. The result of the amino acid analysis and peptide mapping analysis after CNBr cleavage contained no Met. Based on its solubility characteristics at sequential extractions with three acidic solutions (pH 5.5, pH 3.5 and pH 2.6), the histochemically discovered SAPP was believed to be bound to the matrix by a weak ionic bond and covered with mineral. It is suggested that the Met free phosphoprotein is a new protein in the mineralized phase of newborn rat calvaria and participates in the induction of bone mineralization.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Termine, J.D., Belcourt, A.B., Conn, K.M. and Kleinman, H.K.: Mineral and collagen-binding proteins of fetal calf bone. J. Biol. Chem. 256, 10403–10408, 1981.
Hauschka, P.V., Lian, J.B., and Gallop, P.M.: Direct identification of the calcium binding amino acid γ-carboxyglutamate, in mineralized tissue. Proc. Nat. Acad. Sci. USA 72, 3925–3929, 1975.
Prince, P.A., Otsuka, A.S., Poser, J.W., Kristaponis, J., and Raman, N.: Characterization of a γ-carboxyglutamic acid-containing protein from bone. Proc. Nat. Acad. Sci. USA 73, 1447–1451, 1976.
Cohen-Solal, L., Lian, J.B., Kossiva, D. and Glimcher, M.J.: The identification of O-phosphothreonine in the soluble non-collagenous phosphoproteins of bone matrix. FEBS. Lett. 89, 107–110, 1978.
Lee, S.L., and Glimcher, M.J.: Purification, composition, and31P NMR spectroscopic properties of a noncollagenous phosphoprotein isolated from chicken bone matrix. Calcif. Tissue Int. 33, 385–394, 1981.
Uchiyama, A., Suzuki, M., Lefteriou, B. and Glimcher, M.J.: Isolation and chemical characterization of the phosphoproteins of chicken bone matrix: heterogeneity in molecular weight and compositions. Biochemistry 25, 7572–7583, 1986.
Prince, C.W., Oosawa, T., Butler, W.T., Tomana, M., Bhown, A.S., Bhown, M. and Schrohenloher, R.E.: Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone. J. Biol. Chem. 262, 2900–2907, 1987.
Oldberg, A., Franzen, A. and Heinegard, D.: Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. Proc. Nat. Acad. Sci. USA 83, 8819–8823, 1986.
Fisher, L.W., Whitson, S.W., Avioli, L.V. and Termine, J.D.: Matrix sialoprotein of developing bone. J. Biol. Chem. 258, 12723–12727, 1983.
Ashton, B.A., Hohling, H.J., and Triffitt, J.T.: Plasma proteins present in human cortical bone: enrichment of the α2 HS-glycoprotein. Calcif. Tiss. Res. 22, 27–33, 1976.
Triffitt, J.T., Gebauer, U., Ashton, B.A., and Owen, M.E.: Origin of plasma α2 HS-glycoprotein and its accumulation in bone. Nature 262, 226–227, 1976.
Hauschka, P.V.: Osteocalcin structure; Ca2+-dependence of α-helical domains. In The chemistry and biology of mineralized connective tissues (A. Veis ed.), Elsevier, North-Holland, pp. 337–342, 1981.
Fukae, M., Tanabe, T. and Yamada, M.: Non-collagenous proteins of newborn rat calvaria: the possiible mineral and collagen binding proteins. J.B.M.M. 6, 121–131, 1988.
Campbell, K.P., MacLennan, D.H. and Jorgensen, A.O.: Staining of the Ca2+-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, with the cationic carbocyanine dye “Stains-all”. J. Biol. Chem. 258, 11267–11273, 1983.
Maruyama, K., and Nonomura, Y.: High molecular weight calcium binding protein in the microsome of scallop striated muscle. J. Biochem. 96, 859–870, 1984.
Epstein, E.H.,Jr., Scott, R.D., Miller, E.J. and Piez, K.A.: Isolation and characterization of the peptide derived from soluble human and baboon skin collagen after cyanogen bromide cleavage. J. Biol. Chem. 246, 1718–1724, 1971.
Connerty, H.V. and Briggs, A.R.: Determination of serum calcium by means of orthocresolphthalein complexone. Am.J. Clin. Path. 45, 290–296, 1966.
Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680–685, 1970.
Kubota, T., Zhang, Q., Wrana J.L., Ber, R., Aubin, J.E., Butler, W.T. and Sodek, J.: Multiple forms of Sppl (secreted phosphoprotein, osteopontin) synthesized by normal and transformed rat bone cell populations: regulation by TGF-β. Biochem. Biophys. Res. Commun. 162, 1453–1459, 1989.
Green, M. R. and Pastewka, J. V.: Simultaneous differential staining by a cationic carbocyanine dye of nucleic acids, proteins and conjugated proteins. 1. Phosphoproteins. J. Histochem. Cytochem. 22, 767–773, 1974.
Takagi, Y., Fujisawa, R. and Sasaki, S.: Identification of dentin phosphophoryn localization by histochemical stainings. Connect. Tissue Res. 14, 279–292, 1986.
Takagi, Y. and Sasaki, S.: Histological distribution of phosphophoryn in normal and pathological human dentins. J. Oral Pathol. 15, 463–467, 1986.
Craig, A. M., Smith, J. H. and Denhardt, D.T.: Osteopontin, a transformation-associated cell adhesion phosphoprotein, is induced by 12-O-tetradecanoylphorbol 13-acetate in mouse epidermis. J. Biol. Chem. 264, 9682–9689, 1989.
Gorski, J.P. and Shimizu, K.: Isolation of new phosphorylated glycoprotein from mineralized phase of bone that exhibits limited homology to adhesive protein osteopontin. J. Biol. Chem. 263, 15938–15945, 1988.
Butler, W.T., Sato, S., Rahemtulla, F., Prince, C.W., Tomana, M., Bhown, M., DiMuzio, M. T. and Bronkers, A.L.J.J.: Glycoproteins of bone and dentin. In The chemistry and biology of mineralized tissues. (W.T. Butler ed.), Birmingham, ebsco Media, pp. 107–112, 1985.
Sato, S., Rahemtulla, F., Prince, C.W., Tomana, M., and Butler, W.T.: Acidic glycoproteins from bovine compact bone. Connect. Tiss. Res. 14,51–64, 1985.
Butler, W. T., Bhown, M., Tomana, M., Fretwell, B. and Schrohenloher, R.E.: Characterization of a unique dentin glycoprotein. In The chemistry and biology of mineralized connective tissues. (A. Veis ed.), Elsevier, North-Holland, pp, 399–402, 1981.
Author information
Authors and Affiliations
About this article
Cite this article
Fukae, M., Tanabe, T. & Yamada, M. Bone phosphoprotein of newborn rat calvaria detected histochemically with the cationic carbocyanine dye “Stains-all”. J Bone Miner Metab 7, 44–52 (1989). https://doi.org/10.1007/BF02399053
Issue Date:
DOI: https://doi.org/10.1007/BF02399053