| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
When a clonal osteoblastic cell line, MC3T3-E1 cell, was cultured, the cells formed a mineralized matrix after confluence and the protein tyrosine phosphatase (PTP) activity, protein tyrosine kinase activity and levels of some tyrosine phosphorylated protein also increased. As tyrosine phosphorylation is assumed to be important in the cell growth and differentiation, the PTP of the MC3T3-E1 cell was purified and characterized to study the role of PTP on the regulatory mechanism of hard tissue formation. Theree PTPs from the cytosolic fraction of the cells were partially purified. Two were shown to be PTP1B and PTP1D by immunoblotting analysis. The content of the two increased during cell growth and mineralization and they seem to exist as oligomers in the cell. One did not react with commercial anti-PTP antibodies. We purified the PTP to near homogeneity, 4779-fold by several column chromatographic steps. The apparent molecular weight of this PTP was 39 kDa or 33 kDa by sodium dodecyl sulfate polyacrylamido gel electrophoresis and 933 kDa by gel filtration. The optimal pH for PTP activity was acidic, around 6. The activity was inhibited by the usual PTP inhibitors, vanadate, molybdate and zinc but not by serine, threonine phosphatase inhibitor, okadaic acid. The activity was activated by magnesium and inhibited by ethylene diamine tetraacetic acid. These results suggest that the PTPs of MC3T3-E1 cells may participate in the regulation of mineralization and one may be a novel PTP.
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