1993 Fiscal Year Final Research Report Summary
Association of phosphoprotein and collagen with calcification of dentin.
| Project/Area Number |
04671118
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional basic dentistry
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| Research Institution | Hokkaido University |
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Principal Investigator |
FUJISAWA Ryuichi Hokkaido Univ., School of Dentistry, Instructor, 歯学部, 助手 (40190029)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Phosphophoryn / Phosphoprotein / Dentin / Collagen / Calcification / Crosslinking |
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| Research Abstract |
1. Electrostatic interaction of phosphophoryn and collagen
Phosphophoryn, a unique phosphoprotein of dentin, was rabeled with biotin and was subjected to binding assay using collagen as a binding substrate. Phosphophoryn had a significant affinity to collagen. The affinity is thought to be electrostatic, since it was reduced at high ionic strength. Binding site of phosphophoryn in a collagen molecule was determined using CNBr peptides of collagen as binding substrates. The labeled phosphophoryn was preferentially bound to an alpha 1 CB6 peptide, which is corresponding to the C-terminal region.
The labeled phosphophoryn was incubated with reconstituted collagen fibril and was observed with electronmicroscope using avidin-gold as a marker. The phosphophoryn was preferentially bound to hole zones of the fibril.
2. Characterization of phosphophoryn-collagen complex
A part of phosphophoryn is immobilized to collagen in vivo during maturation of dentin.
The phosphophoryn-collagen complex thus formed was solubilized by CNBr digestion ofinsoluble collagen matrix, and purified chromatographically. The complex was composed of phosphophoryn and collagen moieties. These two moieties could not be separated from each other, indicating the covalent nature of the association between them. The complex was reacted with an antibody against alpha 1 CB6 peptide. Proteinase digestion of the complex released a peptide which was derived from alpha 1 CB6 peptide. The covalent binding site of phosphophoryn is deduced to be alpha 1 CB6 peptide.
3. In vitro crosslinking of phosphophoryn with collagen
Phosphophoryn was incubated with insoluble collagen to minic the in vivo crosslinking between these two components. Phosphophoryn was immobilized to collagen with the incubation.
The process was iinhibited by excessive amounts of amines, indicating a possible involvement of dehydroalanine-derived crosslinking.
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[Publications] Zhou, H-Y., Ohnuma, Y., Takita, H., Fujisawa, R., Mizuno, M.and Kuboki, Y.: "Effects of a bone lysine-rich 18K protein on osteoblast-like MC3T3 E1 cells." Biochemical Biophysical Research Communication. 186-13. 1288-1293 (1992)
Description
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