| Co-Investigator(Kenkyū-buntansha) |
AOKI Harumi The Nippon Dental University, Department of Pathology, Assistant Professor, 歯学部, 助教授 (50150925)
YAGISHITA Hisao The Nippon Dental University, Department of Pathology, Assistant, 歯学部, 助手 (50256989)
TAYA Yuji The Nippon Dental University, Department of Pathology, Lecturer, 歯学部, 講師 (30197587)
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| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1996: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1995: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Research Abstract |
1. In the current work, we conducted studies on the molecular structure of the amelogenins by combining multiple techniques in order to gain more concrete information about the structure/function relationship of the major matrix proteins involveld in enamel formation. Techniques used entailed proton nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), small angle x-ray scattering analysis (SAXS), low angle laser light scattering analysis (LALLS), atomic force microscopy (AFM), and immunohistochemistry with epitope-specific antisera. The intact amelogenin protein and its degraded products were isolated and purified from the secretory enamel of slaughtered piglets of 5 to 6 months old. In addition, the 25-residue peptide corresponding to the segment at tha C-terminus was synthesized. The results lead to the following conclusion that : (1) the molecular structure of the intact porcine amelogenin consists of discrete foldin
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g units, i.e., the N-terminal domain containing many aromatic residues, the central domain containing many His and characteristic (QPX)_n repeats, and the hydrophilic domain at the C-terminus : (2) these units have little, if any, interaction with each other. The 5 kD segment at the N-terminus probably has a folded structure, tentatively assigned as beta-sheet structures from CD spectrum, and is more reactive with the cations, particularly calcium. The assumed beta-sheet conformation in the N-terminal region may mediate the intermolecular association of the cleaved fragments of the amelogenin proteins as commonly found at neural pH.The spectral characteristics of the C-terminal domain are similar to those of a random coil conformation. Also the conserved C-terminal domain may direct the molecular structures of the intact amelogenins so as to facilitate the protein-enamel mineral interaction. The plausible conformation of the central domain was characterized by a strong negative ellipticity at 203 nm, similar to that of collagen, although its nature remains to be defined.Based on the information gained in the current work, we propose a tentative structural model of the 20 kD porcine amelogenins, which adopts an elongated bundle structure consisting of one part of beta-sheet and two parts of polyglycine II,interspersed with beta-turn of "loop". It is of interest that the folding sites correspond to the cleavages of the amelogenin in situ, supporting the view that the programd extracellular processing is directly mediated by the conserved primary structure and the resulting folded structure in solution. Less
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