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Immunoblotting studies on artifactual contamination of enamel homogenates by albumin and other proteins

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Abstract

The reason for the presence of albumin and other serum, cytoskeletal, cytosolic, and extracellular matrix proteins in enamel fractions was investigated by immunoblotting using homogenates prepared from freeze-dried and freshly dissected rat incisors, and antibodies capable of resolving at least 1 ng of the primary antigen. The data indicated that most of the 16 antibodies examined in this study reacted with antigens present only within “cell” homogenates (enamel organ cells+adhering labial connective tissue and blood vessels). One exception was rat serum albumin which was detected routinely in enamel homogenates prepared from freshly dissected, wiped incisors but rarely within enamel homogenates prepared from freeze-dried incisors. Another exception was calbindin-D 28 kDa which was consistently found within secretory stage enamel homogenates irrespective of preparative technique. A third exception was enamel proteins (amelogenins) which were enriched in secretory and early maturation stage enamel homogenates compared with cell homogenates and distributed as multiple molecular weight, antigenic bands in enamel homogenates (14–30 kDa), but mostly as a single antigenic band in cell homogenates (near 27 kDa). Overall, the results of this study suggest that developing rat incisor enamel naturally contains few exogenous proteins such as albumin. High concentrations of albumin (or other serum proteins) in crude homogenates, or purified fractions, derive mostly from blood and/or tissue fluids soaking into the enamel during sample preparation. This type of artifact can be avoided by using freeze-dried teeth for biochemical analyses.

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References

  1. Robinson C, Kirkham J (1985) Dynamics of amelogenesis as revealed by protein compositional studies. In: Butler WT (ed) The chemistry and biology of mineralized tissues. Ebsco Media Inc. Birmingham. pp 248–263

    Google Scholar 

  2. Deutsch D (1989) Structure and function of enamel gene products. Anat Rec 224: 189–210

    Google Scholar 

  3. Aoba T, Moreno EC (1991) Structural relationship of amelogenin proteins to their regulatory function of enamel mineralization. In: Sikes CS, Wheeler AP (eds) Surface reactive peptides and polymers. American Chemical Society, Washington, DC, pp 86–106

    Google Scholar 

  4. Nanci A, Smith CE (1992) Development and calcification of enamel. In: Bonucci E (ed) Calcification in biological systems. CRC Press Boca Raton, pp 313–343

    Google Scholar 

  5. 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

    Google Scholar 

  6. Robinson C, Kirkham J, Fincham AG (1989) The enamelin/nonamelogenin problem: a brief review. Connect Tissue Res 22: 93–100

    Google Scholar 

  7. Fincham AG, Hu Y, Lau EC, Slavkin HC, Snead ML (1991) Amelogenin post-secretory processing during biomineralization in the postnatal mouse molar tooth. Arch Oral Biol 36: 305–317

    Google Scholar 

  8. Fincham AG, Moradian-Oldak J, Sarte PE (1994) Massspectrographic analysis of a porcine amelogenin identifies a single phosphorylated locus. Calcif Tissue Int 55: 398–400

    Google Scholar 

  9. Strawich E, Glimcher MJ (1985) Synthesis and degradation in vivo of a phosphoprotein from rat dental enamel. Biochem J 230: 423–433

    Google Scholar 

  10. Smith CE Pompura JR, Borenstein S, Fazel A, Nanci A (1989) Degradation and loss of matrix proteins from developing enamel. Anat Rec 224: 292–316

    Google Scholar 

  11. Aoba T, Shimoda S, Shimokawa H, Inage T (1992) Common epitopes of mammalian amelogenins at the C-terminus and possible functional roles of the corresponding domain in enamel mineralization. Calcif Tissue Int 51: 85–91

    Google Scholar 

  12. Tanabe T, Fukae M, Uchida T, Shimizu M (1992) The localization and characterization of proteinases for the initial cleavage of porcine amelogenin. Calcif Tissue Int 51: 213–217

    Google Scholar 

  13. Fincham AG, Moradian-Oldak J (1993) Amelogenin post-translational modifications: carboxy-terminal processing and the phosphorylation of bovine and porcine “TRAP” and “LRAP” amelogenins. Biochem Biophys Res Commun 197: 248–255

    Google Scholar 

  14. Yamakoshi Y, Tanabe T, Fukae M, Shimizu M (1994) Porcine amelogenins. Calcif Tissue Int 54: 69–75

    Google Scholar 

  15. Moradian-Oldak J, Simmer JP, Sarte PE, Zeichner-David M, Fincham AG (1994) Specific cleavage of a recombinant murine amelogenin at the carboxy-terminal region by a proteinase fraction isolated from developing bovine tooth enamel. Arch Oral Biol 39: 647–656

    Google Scholar 

  16. Zeichner-David M, MacDougall M, Davis A, Vides J, Snead ML, Slavkin HC (1989) Enamelins and amelogenins share the same amino-terminal sequence. Connect Tissue Res 22: 123–129

    Google Scholar 

  17. Tanabe T, Aoba T, Moreno EC, Fukae M, Shimuzu M (1990) Properties of phosphorylated 32 kd nonamelogenin proteins isolated from porcine secretory enamel. Calcif Tissue Int 46:205–215

    Google Scholar 

  18. Deutsch D, Palmon A, Fisher LW, Kolodny N, Termine JD, Young MF (1991) Sequencing of bovine enamelin (“tuftelin”), a novel acidic enamel protein. J Biol Chem 266:16021–16028

    Google Scholar 

  19. Strawich E, Seyer J, Glimcher MJ (1993) Note on tooth enamelins: immuno-identification of two non-amelogenin proteins of developing bovine enamel isolated by affinity chromatography. Further proof that tooth “enamelins” are mainly serum proteins. Connect Tissue Res 29:163–169

    Google Scholar 

  20. Fukae M, Tanabe T, Uchida T, Yamakoshi Y, Shimizu M (1993) Enamelins in the newly formed bovine enamel. Calcif Tissue Int 53:257–261

    Google Scholar 

  21. Limeback H (1987) Isolation and characterization of pig enamelins. Biochem J 243:385–390

    Google Scholar 

  22. Akita H, Fukae M, Shimoda S, Aoba T (1992) Localization of glycosylated matrix proteins in secretory porcine enamel and their possible functional roles in enamel mineralization. Arch Oral Biol 37:953–962

    Google Scholar 

  23. Limeback H, Sakarya H, Chu W, MacKinnon M (1989) Serum albumin and its acid hydrolysis peptides dominate preparations of mineral-bound enamel proteins. J Bone Miner Res 4:235–241

    Google Scholar 

  24. Strawich E, Glimcher MJ (1990) Tooth ‘enamelins’ identified mainly as serum proteins: major ‘enamelin’ is albumin. Eur J Biochem 191:47–56

    Google Scholar 

  25. Menanteau J, Dajean S, Laboux O, Aubry J (1990) Proteins of the mineral compartment of bovine fetal enamel share common antigenic determinants with serum proteins. Calcif Tissue Int 47:251–255

    Google Scholar 

  26. Robinson C, Kirkham J, Brookes SJ, Shore RC (1992) The role of albumin in developing rodent dental enamel: a possible explanation for white spot hypoplasia. J Dent Res 71:1270–1274

    Google Scholar 

  27. Robinson C, Brookes SJ, Kirkham J, Shore RC, Bonass WA (1994) Uptake and metabolism of albumin by rodent incisor enamel in vivo and postmortem: implications for control of mineralization by albumin. Calcif Tissue Int 55:467–472

    Google Scholar 

  28. Couwenhoven RI, Davis C, Snead ML (1989) Mouse ameloblasts do not transcribe the albumin gene. Calcif Tissue Int 45:367–371

    Google Scholar 

  29. Smith CE, Nanci A (1989) A method for sampling the stages of amelogenesis on mandibular rat incisors using the molars as a reference for dissection. Anat Rec 225:257–266

    Google Scholar 

  30. Gotoh Y, Gerstenfeld LC, Glimcher MJ (1990) Identification and characterization of the major chicken bone phosphoprotein—analysis of its synthesis by cultured embryonic chick osteoblasts. Eur J Biochem 187:49–58

    Google Scholar 

  31. Udenfriend S, Stein S, Bohlen P, Dairman W, Leimbgruber W, Weigele M (1972) Fluorescamine, a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178:871–872

    Google Scholar 

  32. Gassmann M, Thömmes P, Weiser T, Hübscher U (1990) Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J 4:2528–2532

    Google Scholar 

  33. Gross E, Witkop B (1962) Nonenzymatic cleavage of peptide bonds: the methionine residues in bovine pancreatic ribonuclease. J Biol Chem 237:1856–1860

    Google Scholar 

  34. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227:680–685

    Google Scholar 

  35. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    Google Scholar 

  36. Kinoshita Y (1979) Incorporation of serum albumin into the developing dentine and enamel matrix in the rabbit incisor. Calcif Tissue Int 29:41–46

    Google Scholar 

  37. McKee MD, Martineau-Doize B, Warshawsky H (1986) Penetration of various molecular-weight proteins into the enamel organ and enamel of the rat incisor. Arch Oral Biol 31:287–296

    Google Scholar 

  38. Lesot H, Smith AJ, Matthews JB, Ruch JV (1988) An extracellular matrix protein of dentine, enamel, and bone shares common antigenic determinants with keratins. Calcif Tissue Int 42:53–57

    Google Scholar 

  39. Robinson C, Lowe NR, Weatherell JA (1975) Amino acid composition, distribution and origin of ‘tuft’ protein in human and bovine dental enamel. Arch Oral Biol 20:29–42

    Google Scholar 

  40. Robinson C, Shore RC, Kirkham J (1989) Tuft protein, its relationship with keratins and the developing enamel matrix. Calcif Tissue Int 44:393–398

    Google Scholar 

  41. Robinson C, Kirkham J, Stonehouse NJ, Shore RC (1989) Extracellular processing of enamel matrix and origin and function of tuft protein. In: Fearnhead RW (ed) Tooth enamel V. Florence Publishers, Yokohama, pp 59–63

    Google Scholar 

  42. Fukae M (1984) Alkaline phosphatase extracted from porcine immature enamel. In: Fearnhead RW, Suga S (eds) Tooth enamel IV. Elsevier Science Publishers. Amsterdam, pp 120–124

    Google Scholar 

  43. Kakei M, Nakahara H, Takeyama H (1991) Immunoblotting study of developing enamel matrix proteins. In: Suga S, Nakahara H (eds) Mechanisms and phylogeny of mineralization in biological systems. Springer-Verlag, Tokyo, pp 95–99

    Google Scholar 

  44. Slavkin HC, Bessem C, Fincham AG, Bringas P Jr, Santos V, Snead ML, Zeichner-David M: (1989) Human and mouse cementum proteins immunologically related to enamel proteins. Biochim Biophys Acta 991:12–18

    Google Scholar 

  45. Inai T, Inai Y, Kurisu K (1993) Immunohistochemical detection of an enamel protein-related epitope in rat bone at an early state of osteogenesis. Histochemistry 99:355–362

    Google Scholar 

  46. Nakamura M, Kindaichi K, Kagayama M (1991) Immunohistochemical and immunochemical detection of a similar epitope in enamel proteins and monocyte-macrophage protein recognized by mouse monoclonal antibody MOMA-2. Arch Oral Biol 36:619–622

    Google Scholar 

  47. Nishikawa S, Kitamura H (1983) Actin filaments in the ameloblast of the rat incisor. Anat Rec 207:245–252

    Google Scholar 

  48. Nishikawa S, Fujiwara K, Kitamura H (1988) Formation of the tooth enamel rod pattern and the cytoskeletal organization in secretory ameloblasts of the rat incisor. Eur J Cell Biol 47:222–232

    Google Scholar 

  49. Goldberg M, Feinberg J, Lecolle S, Kaetzel MA, Rainteau D, Lessard JL, Dedman JR, Weinman S (1991) Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor. Cell Tissue Res 263:81–89

    Google Scholar 

  50. Kasper M, Karsten U, Stosiek P, Moll R (1989) Distribution of intermediate-filament proteins in the human enamel organ: unusually complex pattern of coexpression of cytokeratin polypeptides and vimentin. Differentiation 40:207–214

    Google Scholar 

  51. Takano Y (1979) Cytochemical studies of ameloblasts and the surface layer of enamel of the rat incisor at the maturation stage. Arch Histol Jpn 42:11–32

    Google Scholar 

  52. Nanci A, Zalzal S, Kogaya Y (1993) Cytochemical characterization of basement membranes in the enamel organ of the rat incisor. Histochemistry 99:321–331

    Google Scholar 

  53. Denhardt DT, Guo X (1993) Osteopontin: a protein with diverse functions. FASEB J 7:1475–1482

    Google Scholar 

  54. Kligman D, Hilt DC (1988) The S100 protein family. TIBS 13: 437–443

    Google Scholar 

  55. Heizmann CW, Hunziker W (1991) Intracellular calciumbinding proteins: more sites than insights. TIBS 16:98–103

    Google Scholar 

  56. Taylor AN (1984) Tooth formation and the 28,000-dalton vitamin D-dependent calcium binding protein: an immunocytochemical study. J Histochem Cytochem 32:125–164

    Google Scholar 

  57. Berdal A, Nanci A, Smith CE, Ahluwalia JP, Thomasset M, Cuisinier-Gleizes P, Mathieu H (1991) Differential expression of calbindin-D 28 kDa in rat incisor ameloblasts throughout enamel development. Anat Rec 230:149–163

    Google Scholar 

  58. Nemere I, Leathers VL, Thompson BS, Luben RA, Norman AW (1991) Redistribution of calbindin-D 28k in chick intestine in response to calcium transport. Endocrinology 129:2972–2984

    Google Scholar 

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Authors and Affiliations

  1. Department of Anatomy and Cell Biology, McGill University, 3640 University Street, H3A 2B2, Montreal, Quebec, Canada

    W.-Y. Chen & C. E. Smith

  2. Department of Oral Biology, McGill University, Montreal, Quebec, Canada

    C. E. Smith

  3. Department of Stomatology and Anatomy, Université de Montréal, Montreal, Quebec, Canada

    A. Nanci

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  1. W.-Y. Chen
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  2. A. Nanci
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  3. C. E. Smith
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Chen, WY., Nanci, A. & Smith, C.E. Immunoblotting studies on artifactual contamination of enamel homogenates by albumin and other proteins. Calcif Tissue Int 57, 145–151 (1995). https://doi.org/10.1007/BF00298435

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  • DOI: https://doi.org/10.1007/BF00298435

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