Summary
It was found that significant precipitation occurred immediately after calcium, at a concentration as low as 2 mM, was added to a desalted solution of EDTA extract of adult bovine femur. The maximal yield of the precipitates was observed at a calcium concentration of 30 mM. These precipitates were dissolved in 0.5 M EDTA, desalted, and characterized by Sepharose CL-6B gel filtration chromatography and high performance gelexclusion chromatography. Results revealed that the precipitates were enriched in a 40 K protein and a higher molecular weight fraction as compared with the original extract of bone proteins. The 40 K fraction was isolated and identified as osteonectin, as judged from amino acid analysis, electrophoresis, and immunodetection. The supernatant after calcium-induced precipitation predominantly contained osteocalcin and a 50 K protein that was tentatively identified as α2HS protein. Osteonectin was purified from the calcium-induced precipitates from the EDTA extract of bovine bone. By calcium titration using fluorescence spectrometry, the isolated osteonectin showed high affinity to calcium ions with an apparent dissociation constant (K0.5) of 8×10−7 M. Thus, the use of calcium to separate bone proteins, especially osteonectin, was proved to be a useful technique. In addition, calcium-induced precipitation of osteonectin suggested a possiblein vivo mechanism via which osteonectin might interact with calcium ions and participate in the initial immobilization of calcium to induce the nucleation of calcification in bone tissue.
Similar content being viewed by others
References
Termine JD (1981) Chemical characterization of fetal bone matrix constituents. In: Veis A (ed) The chemistry and biology of mineralized connective tissues. Elsevier North Holland, pp 349–353
Hauschka PV, Lian JB, Gallop PM (1975) Direct identification of the calcium binding amino acid, γ-carboxyglutamate in mineralized tissue. Proc Natl Acad Sci USA 72:3925–3929
Price PA, Otsuka AS, Postner JW, Kristaponis J, Raman N (1976) Characterization of γ-carboxyglutamic acid-containing protein from bone. Proc Natl Acad Sci USA 73:1447–1451
Termine JD, Belcourt AB, Conn KM, Kleinman HK (1981) Mineral and collagen-binding proteins of fetal calf bone. J Biol Chem 256:10403–10408
Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML, Martin GR (1981) Osteonectin, a bone specific protein linking mineral to collagen. Cell 26:99–105
Hauschka PV (1981) Osteocalcin structure: Ca2+-dependence of α-helical domains. In: Veis A (ed) The chemistry and biology of mineralized connective tissues. Elsevier North Holland, pp 337–341
Romberg RW, Werness PG, Loller B, Riggs BL, Mann KG (1985) Isolation and characterization of native adult osteonectin. J Biol Chem 260:2728–2736
Glimcher MJ (1981) On the form and function of bone: from molecules to organ, Wolff's law revisited. In: Veis A (ed) The chemistry and biology of mineralized connective tissues. Elsevier North Holland, pp 617–673
Lee SL, Glimcher MJ (1981) Purification, composition and31P NMR spectroscopic properties of a noncollagenous phosphoprotein isolated from chicken bone matrix. Calcif Tissue Int 33:385–394
Kuboki Y, Fujisawa R, Aoyama K, Sasaki S (1979) Calcium-specific preparation of dentin phosphoprotein: a new method of purification and significance for the mechanism of calcification. J Dent Res 58:1926–1932
Kuboki Y, Fujisawa R, Tsuzaki M, Liu C-F, Sasaki S (1984) Presence of lysinoalanine and histidinoalanine in bovine dentin phosphoprotein. Calcif Tissue Int 36:126–128
Bolander ME, Young MF, Fisher LW, Yamada Y, Termine JD (1988) Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine protease inhibitor (ovomucoid). Proc Natl Acad Sci USA 85:2919–2923
Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfatepoly-acrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203
Sato S, Rahemtulla F, Prince CW, Tomana M, Butler WT (1985) Acidic glycoproteins from bovine compact bone. Connect Tissue Res 14:51–64
Kuboki Y, Takita H, Fujisawa R, Tazaki M, Mizuno M (1987) Histidinoalanine-containing noncollagenous protein in bone. J Dent Res 66: Special issue IADR, AADR Abstract #78, p 116
Ashton BA, Triffitt JT, Herring GM (1974) Isolation and partial characterization of a glycoprotein from bovine cortical bone. Eur J Biochem 45:525–533
Butler WT, Bhown M, Dimuzio MT, Linde A (1981) Noncollagenous proteins of dentin. Isolation and partial characterization of rat dentin proteins and proteoglycans using a three-step preparative method. Coll Res 1:187–199
Rahima M, Veis A (1988) Two classes of dentin phosphophoryns, from wide range of species, contain immunologically cross-reactive epitope regions. Calcif Tissue Int 42:104–112
Cocking-Johnson D, Van Kampen CL, Sauk JJ (1983) Electron-microscopical studies of conformational changes in dentinal phosphophoryn. Coll Rel Res 3:505–510
Linde A (1984) Noncollagenous proteins and proteoglycans in dentinogenesis. In: Linde A (ed) Dentin and Dentinogenesis Vol II. CRC Press, Boca Raton pp 55–92
Uchiyama A, Suzuki M, Lefteriou B, Glimcher MJ (1986) Isolation and chemical characterization of the phosphoproteins of chicken bone matrix: heterogeneity in molecular weight and composition. Biochemistry 25:7572–7583
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kuboki, Y., Takita, H., Komori, T. et al. Separation of bone matrix proteins by calcium-induced precipitation. Calcif Tissue Int 44, 269–277 (1989). https://doi.org/10.1007/BF02553761
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF02553761