Regulatory mechanism of hard tissue formation by tyrosinekinase and phosphatase.

克隆成骨细胞系MC3T3-E1细胞经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均升高。由于酪氨酸磷酸化在细胞生长和分化中起重要作用，我们对MC3T3-E1细胞的PTP进行纯化和表征，研究PTP在硬组织形成的调控机制中的作用。从细胞的细胞质部分纯化了3个ptp。免疫印迹分析显示其中2个为PTP1B和PTP1D。两者的含量在细胞生长和矿化过程中增加，它们似乎以低聚物的形式存在于细胞中。其中一种不与商业抗ptp抗体发生反应。经过几个柱层析步骤，我们纯化的PTP接近均匀性，达到4779倍。经十二烷基硫酸钠聚丙烯酰胺凝胶电泳，该PTP的表观分子量为39 kDa或33 kDa，凝胶过滤的表观分子量为933 kDa。PTP活性的最佳pH值为酸性，约为6。常用的PTP抑制剂钒酸盐、钼酸盐和锌对其活性有抑制作用，而丝氨酸、苏氨酸磷酸酶抑制剂冈田酸对其活性无抑制作用。其活性被镁活化，被乙二胺四乙酸抑制。这些结果提示MC3T3-E1细胞的PTP可能参与矿化调控，可能是一种新的PTP。

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.