Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1979 Jan 1;177(1):81–98. doi: 10.1042/bj1770081

Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken-bone matrix. The presence of O-phosphoserine and O-phosphothreonine in non-collagenous proteins, and their absence from phosphorylated collagen

Lola Cohen-Solal 1,*, Jane B Lian 1, Dora Kossiva 1, Melvin J Glimcher 1,†
PMCID: PMC1186342  PMID: 106848

Abstract

Non-collagenous phosphoproteins, almost all of which can be extracted in EDTA at neutral pH in the presence of proteinase inhibitors, are identified in the matrix of chicken bone, and are therefore not covalently bound to collagen. Similarly, all the peptides containing γ-carboxyglutamic acid are present in the EDTA extract and none in the insoluble residue, confirming that none is covalently linked to chicken bone collagen. However, organic phosphorus is also found to be present in chicken bone collagen, principally in the α2-chains. Of the total protein-bound organic phosphorus present in chicken bone matrix, approx. 80% is associated with the non-collagenous proteins and 20% with collagen. The soluble non-collagenous proteins contain both O-phosphoserine and O-phosphothreonine and these account for essentially of their organic phosphorus content. In contrast, collagen contains neither O-phosphoserine nor O-phosphothreonine. Indeed, no phosphorylated hydroxy amino acid, phosphoamidated amino acid or phosphorylated sugar could be identified in purified components of collagen, which contain approximately four to five atoms of organic phosphorus per molecule of collagen. Peptides containing organic phosphorus were isolated from partial acid hydrolysates and enzymic digests of purified collagen components, which contain an as-yet-unidentified cationic amino acid. These data, the very high concentrations of glutamic acid in the phosphorylated peptides, and the pH-stability of the organic phosphorus moiety in intact collagen chains strongly suggest that at least part of the organic phosphorus in collagen is present as phosphorylated glutamic acid. This would indicate that the two major chemically different protein fractions in chicken bone matrix that contain organic phosphorus may represent two distinct metabolic pools of organic phosphorus under separate biological control.

Full text

PDF
81

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anthony R. S., Spector L. B. Phosphorylated acetate kinase. Its isolation and reactivity. J Biol Chem. 1972 Apr 10;247(7):2120–2125. [PubMed] [Google Scholar]
  2. BOAS N. F. Method for the determination of hexosamines in tissues. J Biol Chem. 1953 Oct;204(2):553–563. [PubMed] [Google Scholar]
  3. Butler W. T., Finch J. E., Jr, Desteno C. V. Chemical character of proteins in rat incisors. Biochim Biophys Acta. 1972 Jan 26;257(1):167–171. doi: 10.1016/0005-2795(72)90266-8. [DOI] [PubMed] [Google Scholar]
  4. Butler W. T., Hall W. T., Richardson W. S. Purification and some properties of the phosphoprotein from rat incisors. Biochim Biophys Acta. 1976 Mar 18;427(1):262–267. doi: 10.1016/0005-2795(76)90302-0. [DOI] [PubMed] [Google Scholar]
  5. CESSI C., PILIEGO F. The determination of amino sugars in the presence of amino acids and glucose. Biochem J. 1960 Dec;77:508–510. doi: 10.1042/bj0770508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carmichael D. J., Chovelon A., Pearson C. H. The composition of the insoluble collagenous matrix of bovine predentine. Calcif Tissue Res. 1975 Jun 18;17(4):263–271. doi: 10.1007/BF02546599. [DOI] [PubMed] [Google Scholar]
  7. Carmichael D. J., Dodd C. M. An investigation of the phosphoprotein of the bovine dentin matrix. Biochim Biophys Acta. 1973 Jul 12;317(1):187–192. doi: 10.1016/0005-2795(73)90211-0. [DOI] [PubMed] [Google Scholar]
  8. Carmichael D. K., Veis A., Wang E. T. Dentin matrix collagen: evidence for a covalently linked phosphoprotein attachment. Calcif Tissue Res. 1971;7(4):331–344. doi: 10.1007/BF02062622. [DOI] [PubMed] [Google Scholar]
  9. ChandraRajan J., Klein L. Determination of inorganic phosphorus in the presence of organic phosphorus and high concentrations of proteins. Anal Biochem. 1976 May 7;72:407–412. doi: 10.1016/0003-2697(76)90548-0. [DOI] [PubMed] [Google Scholar]
  10. Cohen-Solal L., Lian J. B., Kossiva D., Glimcher M. J. The identification of O-phosphothreonine in the soluble non-collagenous phosphoproteins of bone matrix. FEBS Lett. 1978 May 1;89(1):107–110. doi: 10.1016/0014-5793(78)80533-x. [DOI] [PubMed] [Google Scholar]
  11. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  12. DRYER R. L., TAMMES A. R., ROUTH J. I. The determination of phosphorus and phosphatase with N-phenyl-p-phenylenediamine. J Biol Chem. 1957 Mar;225(1):177–183. [PubMed] [Google Scholar]
  13. Dickson I. R., Dimuzio M. T., Volpin D., Ananthanarayanan S., Veis A. The extraction of phosphoproteins from bovine dentin. Calcif Tissue Res. 1975 Nov 24;19(1):51–61. doi: 10.1007/BF02563990. [DOI] [PubMed] [Google Scholar]
  14. Edelman M., Hirsch C. A., Hiatt H. H., Fox M. Apparent changes in mouse liver DNA content due to interference by non-DNA diphenylamine-reacting cytoplasmic material. Biochim Biophys Acta. 1969 Mar 18;179(1):172–178. doi: 10.1016/0005-2787(69)90133-6. [DOI] [PubMed] [Google Scholar]
  15. FRANCOIS P., HERMAN H. [The fundamental mineral compound of calcified tissues. II. Bone salts containing a calcium phosphate different from hydroxylapatite]. Bull Soc Chim Biol (Paris) 1961;43:643–649. [PubMed] [Google Scholar]
  16. Furthmayr H., Timpl R. Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem. 1971 Jun;41(2):510–516. doi: 10.1016/0003-2697(71)90173-4. [DOI] [PubMed] [Google Scholar]
  17. GLIMCHER M. J., FRANCOIS C. J., RICHARDS L., KRANE S. M. THE PRESENCE OF ORGANIC PHOSPHORUS IN COLLAGENS AND GELATINS. Biochim Biophys Acta. 1964 Dec 9;93:585–602. doi: 10.1016/0304-4165(64)90342-3. [DOI] [PubMed] [Google Scholar]
  18. GLIMCHER M. J., KRANE S. M. THE IDENTIFICATION OF SERINE PHOSPHATE IN ENAMEL PROTEINS. Biochim Biophys Acta. 1964 Sep 4;90:477–483. doi: 10.1016/0304-4165(64)90227-2. [DOI] [PubMed] [Google Scholar]
  19. GLIMCHER M. J., KRANE S. M. THE INCORPORATION OF RADIOACTIVE INORGANIC ORTHOPHOSPHATE AS ORGANIC PHOSPHATE BY COLLAGEN FIBRILS IN VITRO. Biochemistry. 1964 Feb;3:195–202. doi: 10.1021/bi00890a010. [DOI] [PubMed] [Google Scholar]
  20. GRANT R. A. ESTIMATION OF HYDROXYPROLINE BY THE AUTOANALYSER. J Clin Pathol. 1964 Nov;17:685–686. doi: 10.1136/jcp.17.6.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gilmcher M. J., Katz E. P. The organization of collagen in bone: the role of noncovalent bonds in the relative insolubility of bone collagen. J Ultrastruct Res. 1965 Jun;12(5):705–729. doi: 10.1016/s0022-5320(65)80057-0. [DOI] [PubMed] [Google Scholar]
  22. Glimcher M. J., Levine P. T. Studies of the proteins, peptides and free amino acids of mature bovine enamel. Biochem J. 1966 Mar;98(3):742–753. doi: 10.1042/bj0980742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Green M. R., Pastewka J. V., Peacock A. C. Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye. Anal Biochem. 1973 Nov;56(1):43–51. doi: 10.1016/0003-2697(73)90167-x. [DOI] [PubMed] [Google Scholar]
  24. HERMAN H., DALLEMAGNE M. J. The main mineral constituent of bone and teeth. Arch Oral Biol. 1961 Nov;5:137–144. doi: 10.1016/0003-9969(61)90007-3. [DOI] [PubMed] [Google Scholar]
  25. Hauschka P. V., Lian J. B., Gallop P. M. Direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3925–3929. doi: 10.1073/pnas.72.10.3925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hauschka P. V. Quantitative determination of gamma-carboxyglutamic acid in proteins. Anal Biochem. 1977 May 15;80(1):212–223. doi: 10.1016/0003-2697(77)90640-6. [DOI] [PubMed] [Google Scholar]
  27. Holbrook I. B., Leaver A. G. Glycoproteins in the dentine of human deciduous teeth. Arch Oral Biol. 1976;21(9):509–512. doi: 10.1016/0003-9969(76)90015-7. [DOI] [PubMed] [Google Scholar]
  28. Höhling H. J., Ashton B. A., Köster H. D. Quantitative electron microscopic investigations of mineral nucleation in collagen. Cell Tissue Res. 1974 Mar 29;148(1):11–26. doi: 10.1007/BF00224315. [DOI] [PubMed] [Google Scholar]
  29. Höhling H. J., Kreilos R., Neubauer G., Boyde A. Electron microscopy and electron microscopical measurements of collagen mineralization in hard tissues. Z Zellforsch Mikrosk Anat. 1971;122(1):36–52. doi: 10.1007/BF00936115. [DOI] [PubMed] [Google Scholar]
  30. Jones I. L., Leaver A. G. Studies on the minor components of the organic matrix of human dentine. Arch Oral Biol. 1974 May;19(5):371–380. doi: 10.1016/0003-9969(74)90178-2. [DOI] [PubMed] [Google Scholar]
  31. Leaver A. G., Holbrook I. B., Jones I. L., Thomas M., Sheil L. Components of the organic matrices of bone and dentine isolated only after digestion with collagenase. Arch Oral Biol. 1975 Mar;20(3):211–216. doi: 10.1016/0003-9969(75)90011-4. [DOI] [PubMed] [Google Scholar]
  32. Leaver A. G., Triffitt J. T., Holbrook I. B. Newer knowledge of non-collagenous protein in dentin and cortical bone matrix. Clin Orthop Relat Res. 1975 Jul-Aug;(110):269–292. doi: 10.1097/00003086-197507000-00039. [DOI] [PubMed] [Google Scholar]
  33. 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. 1967 Feb;12(2):311–313. doi: 10.1016/0003-9969(67)90053-2. [DOI] [PubMed] [Google Scholar]
  34. Li S. T., Katz E. P. On the state of anionic groups of demineralized matrices of bone and dentine. Calcif Tissue Res. 1977 Feb 11;22(3):275–284. doi: 10.1007/BF02010366. [DOI] [PubMed] [Google Scholar]
  35. Menanteau J., Pieri J., Kerebel B. Etude d'une fraction protéique non collagénique de la dentine en voie de minéralisation. J Biol Buccale. 1977 Mar;5(1):23–29. [PubMed] [Google Scholar]
  36. Miller G. L. Tolerance following immunity, with loss of serum specificity, in adult rabbits injected repeatedly with HeLa cells. J Immunol. 1971 Jun;106(6):1654–1659. [PubMed] [Google Scholar]
  37. Mårdh S., Ljungström O., Högstedt S., Zetterqvist O. Studies on a rat-liver cell-sap protein yielding 3-[32P]-phosphohistidine after incubation with [32P]ATP and alkaline hydrolysis. Identification of the protein as ATP citrate lyase. Biochim Biophys Acta. 1971 Dec 28;251(3):419–426. doi: 10.1016/0005-2795(71)90131-0. [DOI] [PubMed] [Google Scholar]
  38. NAGAI Y., GROSS J., PIEZ K. A. DISC ELECTROPHORESIS OF COLLAGEN COMPONENTS. Ann N Y Acad Sci. 1964 Dec 28;121:494–500. doi: 10.1111/j.1749-6632.1964.tb14221.x. [DOI] [PubMed] [Google Scholar]
  39. Nagano K., Kanazawa T., Mizuno N., Tashima Y., Nakao T., Nakao M. Some acyl phosphate-like properties of P32-labeled sodium-potassium-activated adenosine triphosphatase. Biochem Biophys Res Commun. 1965 Jun 9;19(6):759–764. doi: 10.1016/0006-291x(65)90324-4. [DOI] [PubMed] [Google Scholar]
  40. ORNSTEIN L. DISC ELECTROPHORESIS. I. BACKGROUND AND THEORY. Ann N Y Acad Sci. 1964 Dec 28;121:321–349. doi: 10.1111/j.1749-6632.1964.tb14207.x. [DOI] [PubMed] [Google Scholar]
  41. 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. 1977 Jul 15;79(2):276–280. doi: 10.1016/0014-5793(77)80802-8. [DOI] [PubMed] [Google Scholar]
  42. Pieri J., Menanteau J., Kerebel B. Caractérisation des protéines non collagéniques de la dentine en voie de minéralisation. C R Acad Sci Hebd Seances Acad Sci D. 1975 Dec 15;281(23):1905–1908. [PubMed] [Google Scholar]
  43. Plimmer R. H. Esters of phosphoric acid: Phosphoryl hydroxyamino-acids. Biochem J. 1941 Apr;35(4):461–469. doi: 10.1042/bj0350461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Price P. A., Otsuka A. A., Poser J. W., Kristaponis J., Raman N. Characterization of a gamma-carboxyglutamic acid-containing protein from bone. Proc Natl Acad Sci U S A. 1976 May;73(5):1447–1451. doi: 10.1073/pnas.73.5.1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. ROBINSON R. A., WATSON M. L. Collagen-crystal relationships in bone as seen in the electron microscope. Anat Rec. 1952 Nov;114(3):383–409. doi: 10.1002/ar.1091140302. [DOI] [PubMed] [Google Scholar]
  46. ROBINSON R. A., WATSON M. L. Crystalcollagen relationships in bone as observed in the electron microscope. III. Crystal and collagen morphology as a function of age. Ann N Y Acad Sci. 1955 Apr 27;60(5):596–628. doi: 10.1111/j.1749-6632.1955.tb40054.x. [DOI] [PubMed] [Google Scholar]
  47. ROE J. H. The determination of sugar in blood and spinal fluid with anthrone reagent. J Biol Chem. 1955 Jan;212(1):335–343. [PubMed] [Google Scholar]
  48. SCHLUETER R. J., VEIS A. THE MACROMOLECULAR ORGANIZATION OF DENTINE MATRIX COLLAGEN. II. PERIODATE DEGRADATION AND CARBOHYDRATE CROSS-LINKING. Biochemistry. 1964 Nov;3:1657–1665. doi: 10.1021/bi00899a010. [DOI] [PubMed] [Google Scholar]
  49. Sakai T., Gross J. Some properties of the products of reaction of tadpole collagenase with collagen. Biochemistry. 1967 Feb;6(2):518–528. doi: 10.1021/bi00854a021. [DOI] [PubMed] [Google Scholar]
  50. Seyer J. M., Glimcher M. J. Evidence for the presence of numerous protein components in immature bovine dental enamel. Calcif Tissue Res. 1977 Dec 29;24(3):253–257. doi: 10.1007/BF02223325. [DOI] [PubMed] [Google Scholar]
  51. 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 Acta. 1977 Aug 23;493(2):441–451. doi: 10.1016/0005-2795(77)90200-8. [DOI] [PubMed] [Google Scholar]
  52. Seyer J. M., Glimcher M. J. The isolation of phosphorylated polypeptide components of the organic matrix of embryonic bovine enamel. Biochim Biophys Acta. 1971 Apr 27;236(1):279–291. doi: 10.1016/0005-2795(71)90177-2. [DOI] [PubMed] [Google Scholar]
  53. Seyer J., Glimcher M. J. The amino acid sequence of two O-phosphoserine containing tripeptides isolated from the organic matrix of embryonic bovine enamel. Biochim Biophys Acta. 1969 Jul 1;181(2):410–418. doi: 10.1016/0005-2795(69)90274-8. [DOI] [PubMed] [Google Scholar]
  54. Shuttleworth A., Veis A. The isolation of anionic phosphoproteins from bovine cortical bone via the periodate solubilization of bone collagen. Biochim Biophys Acta. 1972 Feb 29;257(2):414–420. doi: 10.1016/0005-2795(72)90294-2. [DOI] [PubMed] [Google Scholar]
  55. Spector A. R., Glimcher M. J. The identification of O-phosphoserine in the soluble anionic phosphoproteins of bone. Biochim Biophys Acta. 1973 Apr 20;303(2):360–362. doi: 10.1016/0005-2795(73)90367-x. [DOI] [PubMed] [Google Scholar]
  56. Thomas M., Leaver A. G. The less-acidic glycoproteins of the organic matrix of human dentine. Arch Oral Biol. 1977;22(8-9):545–549. doi: 10.1016/0003-9969(77)90053-x. [DOI] [PubMed] [Google Scholar]
  57. VEIS A., SCHLUETER R. J. THE MACROMOLECULAR ORGANIZATION OF DENTINE MATRIX COLLAGEN. I. CHARACTERIZATION OF DENTINE COLLAGEN. Biochemistry. 1964 Nov;3:1650–1657. doi: 10.1021/bi00899a009. [DOI] [PubMed] [Google Scholar]
  58. Veis A., Perry A. The phosphoprotein of the dentin matrix. Biochemistry. 1967 Aug;6(8):2409–2416. doi: 10.1021/bi00860a017. [DOI] [PubMed] [Google Scholar]
  59. Veis A., Spector A. R., Carmichael D. J. The organization and polymerization of bone and dentin collagens. Clin Orthop Relat Res. 1969 Sep-Oct;66:188–211. [PubMed] [Google Scholar]
  60. Veis A., Spector A. R., Zamoscianyk H. The isolation of an EDTA-soluble phosphoprotein from mineralizing bovine dentin. Biochim Biophys Acta. 1972 Feb 29;257(2):404–413. doi: 10.1016/0005-2795(72)90293-0. [DOI] [PubMed] [Google Scholar]
  61. Walsh C. T., Jr, Hildebrand J. G., Spector L. B. Succinyl phosphate. Its nonenzymatic hydrolysis and reaction with coenzyme A. J Biol Chem. 1970 Nov 10;245(21):5699–5708. [PubMed] [Google Scholar]
  62. Weinstock M., Leblond C. P. Radioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentin of the rat incisor. J Cell Biol. 1973 Mar;56(3):838–845. doi: 10.1083/jcb.56.3.838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zetterqvist O., Engström L. Isolation of N-e-[32P]phosphoryl-lysine from rat-liver cell sap after incubation with [32P]adenosine triphosphate. Biochim Biophys Acta. 1967 Aug 29;141(3):523–532. doi: 10.1016/0304-4165(67)90181-x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES