1987 Fiscal Year Final Research Report Summary
Tissue distribution and purification of Phosphophoryn an approach using the monoclonal antibody
| Project/Area Number |
61480391
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional basic dentistry
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| Research Institution | Kagoshima University |
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Principal Investigator |
NAKAMA Takako Assistant, Kagoshima University Dental School, 歯学部, 助手 (60128444)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAMURA Osamu Assistnat, Kagoshima University Dental School, 歯学部, 助手 (70128445)
DAIKUHARA Yasushi Professor, Kagoshima University Dental School, 歯学部, 教授 (40028733)
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| Project Period (FY) |
1986 – 1987
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| Keywords | Dentin / bone / phosphophoryn / calcification / monoclonal antibody / 骨由来非コラーゲン性タンパク質 |
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| Research Abstract |
We Previously raised a monoclonal antibody against phoshpophoryn from fetal bovine dentin. During studies on the tissue specificity of this sntibody, we unexpectedly found that newly formed long bone tissues were stained immunohistochemically with the antibody. Further study on this cross-reactivity of the antibody was the main purpose of this project.
Osteoblasts, Osteocytes and the bone matrix deposited on the surface of the cartilage in decalcified and nondecalcified sections of the epiphyseal portion of the long bone were stained immunohistochemically with the monoclonal antibody against phosphophoryn. However, Chondrocytes and the various zones of the epiphyseal cartilage matrix were not stained. In a control sections, none of these structures were stained. No difference in staining properties was observed in mumbranous bone from the cartilagineus bone. Large cuboidal cells those were thought to be preosteoblasts were also stained. These findings suggest that a protein(s) which was
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recognized by the antibody may be involved in the calcification process on the bone.
Next we tried to isolate the protein(s) from fresh fetal bovine long bones. Noncollagenous bone proteins were extracted from the tissue by treatment with 4M guanidine-HC1 (pH 7.4) containing various inhibitors of protease, decalcified with 0.5 M EDTA and then with 0.1M formic acid. The extract was applied to a column of Q-Sepharose and then material(s) which was reactive to the antibody by ELISA was eluted with linear gradient of NaCl. This material(s) showed two bands by SDS-PAGE with silver staining and the molecular weights were estimated to be 71k and 63k. After Western blotting, these two bands were also stained by ELISA. The material not treated with SDS, however, Showed only one peak at molecular weight of 66k on a column of Sephacryl S-300. Attempts to separate these two proteins by various methods including HPLC have been failed. Therefore, we extracted the proten directly from a band n the gel of SDS-PAGE. Two final preparations showed only one band of either 71k and 63k by SDS-PAGE with silver staining. Characterization of these two noncollagenous proteins from fetal bovine bone is now under investigation. Less
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Research Products
(4 results)
