Studies on the enzyme responsible for tissue degradation of a periodontopathogenic bacterium Porphyromonas gingivalis
Project/Area Number |
06671842
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Research Category |
Grant-in-Aid for General Scientific Research (C)
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Allocation Type | Single-year Grants |
Research Field |
Morphological basic dentistry
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Research Institution | Matsumoto Dental College, Faculty of Dentistry |
Principal Investigator |
FUJIMURA Setsuo Matsumoto Dental College, Faculty of Dentistry, Assistant Professor, 歯学部, 助教授 (40045505)
|
Co-Investigator(Kenkyū-buntansha) |
NAKAMURA Takeshi Matsumoto Dental College, Faculty of Dentistry, Professor, 歯学部, 教授 (60064656)
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Project Period (FY) |
1994 – 1995
|
Project Status |
Completed (Fiscal Year 1995)
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Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,100,000 (Direct Cost: ¥1,100,000)
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Keywords | P.gingivalis / Protease / Purification / Properties / Hemoglobin / Envelope / Binding / Binding-protein / P. gingivalis / 歯周病原菌 / 蛋白分解酵素 / p.gingivalis |
Research Abstract |
A lysin-specific protease hydrolysing peptide bonds at the carboxyl side of lysine residues in Porphyromonas gingivalis was purified from culture supernatant by a combination of ion exchange chromatography, gel filtration, and affinity chromatography. The molecular mass was 48 kDa and pI value was 7.3. The enzyme hydrolyzed the peptide bonds at the carboxyl side of lysine residues in synthetic substrates and natural proteins. P.gingivalis was found to bind to several hemoproteins and the binding properties of the envelope to hemoglobin were investigated. Maximum amount of hemoglobin bound to 1 mg of the envelope was 58mug. No significant binding was observed at 4゚C.Heating of the envelope at 70゚C for 15 min resulted in complete loss of the binding activity. The envelope saturated with hemoglobin could no longer bind to other hemoproteins tested, indicating that binding site for the hemoproteins are common. The binding activity of the envelope to hemoglobin was examined over a wide range of pH from 4.5 to 9.0. The binding activity in low pH buffers was much higher than that at high pH,the optimum pH for the binding was found at 4.5 and 5.0. Since the bound hemoglobin to the envelope was found to dissociate in the pH 8.5 or 9.0 buffers, the binding is reversible. We supposed that hemoglobin-binding protein (HbBP) responsible for the binding to hemoglobin exists in the envelope, and confirmed its presence by dot blot assay using peroxidase-conjugated hemoglobin. Then we isolated HbBP from the detergent-solubilized materials of the envelope using affinity chromatography. The molecular mass of HbBP was 19 kDa and pI value was 4.3.
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Report
(3 results)
Research Products
(7 results)