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Regulatory mechanism of hard tissue formation by tyrosinekinase and phosphatase.

酪氨酸激酶和磷酸酶对硬组织形成的调节机制。

基本信息

批准号：
07672006
负责人：
SUZUKI Kuniaki
金额：
$ 1.6万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672006/
关键词：
Tyrosine Kinase Tyrosine phosphatase MC3T3-E1 cell hard tissue formation PTP1B PTP1D Tyrosine phosphorylation

项目摘要

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.

培养成骨细胞系MC 3 T3-E1细胞，细胞融合后形成矿化基质，蛋白酪氨酸磷酸酶（PTP）活性、蛋白酪氨酸激酶活性及部分酪氨酸磷酸化蛋白水平升高。由于酪氨酸磷酸化被认为是重要的细胞生长和分化，PTP的MC 3 T3-E1细胞的纯化和表征，研究PTP的硬组织形成的调节机制的作用。来自细胞的胞质级分的三种PTP被部分纯化。经免疫印迹分析，其中两个为PTP 1B和PTP 1D。在细胞生长和矿化过程中，两者的含量增加，并且它们似乎以低聚物的形式存在于细胞中。一个不与商业抗PTP抗体反应。我们通过几个柱层析步骤将PTP纯化至接近均一性，4779倍。该PTP的表观分子量为39 kDa或33 kDa的十二烷基硫酸钠聚丙烯酰胺凝胶电泳和933 kDa的凝胶过滤。PTP活性的最适pH为酸性，约为6。该活性被通常的PTP抑制剂钒酸盐、EDTA和锌抑制，但不被丝氨酸、苏氨酸磷酸酶抑制剂冈田酸抑制。镁对酶有激活作用，乙二胺四乙酸对酶有抑制作用。这些结果表明，MC 3 T3-E1细胞的PTP可能参与了矿化的调节，其中一个可能是一种新的PTP。