| Project/Area Number |
12670258
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Kagawa Medical School |
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Principal Investigator |
MATSUSHITA Osamu Kagawa Medical University, Microbiology, assistant Professor, 医学部, 助教授 (00209537)
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| Co-Investigator(Kenkyū-buntansha) |
NISHI Nozomu Kagawa Medical University, Endocrinology, research associate, 医学部, 助手 (10145047)
片山 誠一 香川医科大学, 医学部, 助手 (70169473)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Clostridium / collagenase / collagen-binding domain / beta-sandwitch / collagenous peptide |
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| Research Abstract |
Tandem collagen-binding domains (CBD's) are present at the C-terminus of Clostridium histicum class I collagenase. Three-dimesional structure of the domain was determined in the presence and abscence of Ca^<2+> ion. Addition of the ion altered conformation of the N-terminal linker peptide from an alpha-helix to beta-sheets, which stabilizes the beta-sandwich domain structure and increases the substrate affinity. (Joint project with Dr. Joshua Sakon et al. University of Arkansas, U. S. A.) in order to investigate the mode of substrate binding, mutated CBD's were constructed, where various surface-oriented amino acid residues are altered. By surface plasmon resonance using a sensor chip with an immobilized collagenous peptide, G(POG)_8, we determined their binding constants against this artificial substrate. This expriment showed that a hydrophobic surface of the sandwitch plays a key role for the substrate binding.
Binding of CBD against various types of collagen was studied by immunohistochemistry. Light and electron microscopic observation was performed after allowing CBD to bind to prefixed collagen-rich tissues, i.e. kidney, cartilage and aorta. CBD bound to all these tissues, but with no periodicity. CBD also bound to various types of collagen in vitro. These results suggested that CBD recognizes its triple helical confomation.
We purified collagenases from three gelatinolytic Clostridia, and cloned their structural genes. Comparison of the deduced sequences revealed that they possess unique segmental structure. We could observe the dynamic rearrangements of the enzyme strucure by comparing the primary sequence of various enzymes.
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