Skip to main content
SpringerLink
Log in

Identification of the noncollagenous proteins of bovine bone by two-dimensional gel electrophoresis

  • Laboratory Investigations
  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Summary

We have characterized the noncollagenous proteins of bovine bone using two high-resolution gel electrophoretic techniques. Proteins were extracted from bone tissue by extended dialysis against 0.5 M EDTA. In some cases, a preextraction was done in guanidine HCl. Bovine plasma was also examined to identify the proteins in bone that might also be present in blood. We have scored 160 major, reproducible spots on our standard preparation bone map. These comprise about 40 individual protein groups. There are many more minor spots present which puts the total number present over 200. Of these groups, 15 are not present on plasma maps. Bone proteins identified in this way include actin, bone Gla-protein, and osteonectin. The remainder are unknown. Bone Gla-protein is present in bovine bone in four isoelectric forms, pI=3.95−4.50. Electroblotting analysis of EDTA and guanidine HCl extracted material failed to reveal any higher molecular weight immunologically reactive species. The plasma proteins found in bone include, but are not limited to albumin, apo A-I lipoprotein, IgG, IgM, transferrin, α-2-HS-glycoprotein, and hemoglobin. Extraction with guanidine HCl plus EDTA significantly enriches the yield for the nonplasma proteins but does not appear to extract any additional bone proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Herring GM (1968) Studies on the protein-bound chondroitin sulfate of bovine cortical bone. Biochem J 107:41–49

    CAS  PubMed  Google Scholar 

  2. Spector AR, Glimcher MJ (1972) The extraction and characterization of soluble anionic phosphoproteins from bone. Biochim Biophys Acta 263:593–603

    CAS  Google Scholar 

  3. Triffitt JT, Owen M (1973) Studies of bone matrix glycoproteins: incorporation of [1-14C]glucosamine and plasma [14C]glycoprotein into rabbit cortical bone. Biochem J 136:125–134

    CAS  PubMed  Google Scholar 

  4. Shuttleworth A, Veis A (1972) The isolation of anionic phosphoproteins from bovine cortical bone via the periodate solubilization of bone collagen. Biochim Biophys Acta 257:414–420

    CAS  PubMed  Google Scholar 

  5. Dickson I (1974) The composition and antigenicity of sheep cortical bone matrix proteins. Calcif Tissue Res 16:321–333

    Article  CAS  PubMed  Google Scholar 

  6. Ashton BA, Höhling H-J, Triffitt JT (1976) Plasma proteins present in human cortical bone: enrichment of the α2HS-glycoprotein Calcif Tissue Res 22:27–33

    Article  CAS  Google Scholar 

  7. Leaver AG, Triffitt JT, Holbrook IB (1975) Newer knowledge of non-collagenous protein in dentin and cortical bone matrix. Clin Orthop 110:269–292

    CAS  PubMed  Google Scholar 

  8. Urist MR, Mikulski A, Lietze A (1979) Solubilized and insolubilized bone morphogenetic protein. Proc Natl Acad Sci USA 76:1828–1832

    CAS  PubMed  Google Scholar 

  9. Lee S, Glimcher MJ (1981) Purification, composition, and31P NMR spectroscopic properties of a noncollagenous phosphoprotein isolated from chicken bone matrix. Calcif Tissue Int 33:385–394

    CAS  PubMed  Google Scholar 

  10. Mbuyi J-M, Dequeker J, Bloemmen F, Stevens E (1982) Plasma proteins in human cortical bone: enrichment of α2HS-glycoprotein, α1acid-glycoprotein, and IgE. Calcif Tissue Int 34:229–231

    CAS  PubMed  Google Scholar 

  11. Triffitt JT, Owen M (1977) Preliminary studies on the binding of plasma albumin to bone tissue. Calcif Tissue Res 23:303–305

    Article  CAS  PubMed  Google Scholar 

  12. Triffitt JT, Grebauer U, Ashton BA, Owen ME, Reynolds JJ (1976) Origin of plasma α2HS-glycoprotein and its accumulation in bone Nature 262:226–227

    Article  CAS  PubMed  Google Scholar 

  13. Triffitt JT, Owen ME, Ashton BA, Wilson JM (1978) Plasma disappearance of rabbit2HS-glycoprotein and its uptake by bone tissue. Calcif Tissue Res 26:155–161

    Article  CAS  PubMed  Google Scholar 

  14. 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

    CAS  PubMed  Google Scholar 

  15. Price PA, Otsuka AS, Poser JW, Kristaponis J, Raman N (1976) Characterization of a γ-carboxyglutamic acid-containing protein from bone. Proc Natl Acad Sci USA 73:1447–1451

    CAS  PubMed  Google Scholar 

  16. Hauschka PV, Frenkel J, DeMuth R, Gundberg CM (1983) Presence of osteocalcin and related higher molecular weight 4-carboxyglutamic acid-containing proteins in developing bone. J Biol Chem 258:176–182

    CAS  PubMed  Google Scholar 

  17. Termine JD, Belcourt AB, Conn KM, Kleinman HK (1981) Mineral and collagen-binding proteins of fetal calf bone. J Biol Chem 256:10403–10408

    CAS  PubMed  Google Scholar 

  18. Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey JL, Martin GR (1981) Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26:99–105

    Article  CAS  PubMed  Google Scholar 

  19. Farley JR, Baylink DJ (1982) Purification of a skeletal growth factor from human bone. Biochemistry 21:3502–3507

    Article  CAS  PubMed  Google Scholar 

  20. Termine JD, Belcourt AB, Christner PJ, Conn KM, Nylen MU (1980) Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel. J Biol Chem 255:9760–9768

    CAS  PubMed  Google Scholar 

  21. Price PA, Nishimoto SK (1980) Radioimmunoassay for the vitamin K-dependent protein of bone and its discovery in plasma. Proc Natl Acad Sci USA 77:2234–2238

    CAS  PubMed  Google Scholar 

  22. Greenwood FC, Hunter WM, Glover JS (1963) The preparation of131I-labelled human growth hormone of high specific radioactivity. Biochem J 89:114–123

    CAS  PubMed  Google Scholar 

  23. Anderson L, Anderson NG (1977) High resolution two-dimensional electrophoresis of human plasma proteins. Proc Natl Acad Sci USA 74:5421–5425

    CAS  PubMed  Google Scholar 

  24. Tracy RP, Currie RM, Young DS (1982) Two-dimensional gel electrophoresis of serum specimens from a normal population. Clin Chem 28:890–899

    CAS  PubMed  Google Scholar 

  25. Tracy RP, Currie RM, Young DS (1982) Reproducibility and quality assurance of two-dimensional gel electrophoresis of serum specimens. Clin Chem 28:908–914

    CAS  PubMed  Google Scholar 

  26. Tracy RP, Currie RM, Hill HD, Young DS (1983) Methods of two-dimensional gel electrophoresis for the clinical laboratory. Prot Biol Fluids 30:581–586

    Google Scholar 

  27. Oakley BR, Kirsch DR, Morris NR (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105:361–363

    Article  CAS  PubMed  Google Scholar 

  28. Tracy RP, Wold LE, Currie RM, Young DS (1982) Patterns for normal colon mucosa and colon adenocarcinoma compared by two-dimensional gel electrophoresis. Clin Chem 28:915–919

    CAS  PubMed  Google Scholar 

  29. Anderson NL, Nance SL, Pearson T, Anderson NG (1982) Specific antiserum staining of two-dimensional electrophoretic patterns of human plasma proteins immobilized on nitrocellulose. Electrophoresis 3:135–142

    Article  CAS  Google Scholar 

  30. Blumenthal NC, Betts F, Posner AS (1975) Effect of carbonate and biological macromolecules on formation and properties of hydroxyapatite. Calcif Tissue Res 18:81–90

    CAS  PubMed  Google Scholar 

  31. Hauschka PV, Gallop PM (1977) Purification and calcium-binding properties of osteocalcin, the γ-carboxyglutamate-containing protein of bone. In: Wasserman RH, Corradino RA, Carafoli E, Kretsinger RH, MacLennan DH, Siegel FL (eds) Calcium binding proteins and calcium function. Elsevier/North Holland Biomedical Press, New York, Amsterdam, Oxford, p 338

    Google Scholar 

  32. Nishimoto SK, Price PA (1980) Secretion of the vitamin K-dependent protein of bone by rat osteosarcoma cells. J Biol Chem 255:6579–6583

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Internal Medicine, The Section of Hematology Research and the Endocrine Research Unit, USA

    Pierre D. Delmas, Russell P. Tracy, B. Lawrence Riggs & Kenneth G. Mann

  2. Department of Laboratory Medicine, Mayo Clinic/Foundation, 55905, Rochester, Minnesota

    Pierre D. Delmas, Russell P. Tracy, B. Lawrence Riggs & Kenneth G. Mann

Authors
  1. Pierre D. Delmas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Russell P. Tracy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B. Lawrence Riggs
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kenneth G. Mann
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Delmas, P.D., Tracy, R.P., Riggs, B.L. et al. Identification of the noncollagenous proteins of bovine bone by two-dimensional gel electrophoresis. Calcif Tissue Int 36, 308–316 (1984). https://doi.org/10.1007/BF02405335

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02405335

Key Words

Search

Navigation