Molecular characteristics and physiological roles of phosphatases in calcifying matrix vesicles.

在钙化初期导致基质囊泡(MV)中磷灰石形成的分子过程，迄今尚未从囊泡内磷酸酶的功能行为中得到了解。两种类型的MV以不同的钙化方式参与了该项目的推进。(1)软骨骨化过程中的MV：经Percoll密度梯度离心法分离后的小牛骨骺软骨MV显示出最高的碱性磷酸酶(ALP)比活性。ALP的磷酸转移酶活性被解释为在MV中或由MV引起的钙化过程中的一种重要功能。从酸性磷酸酶(ACP)/碱性磷酸酶(ALP)活性不固定的比率推测MV的明显异质性，不能将ACP归因于一种独特的囊泡酶，尽管非溶菌体ACP可能存在于MV中。(2)膜内成骨过程中的MV：观察小鼠颅骨成骨克隆MC3T3-EI培养细胞的MV形成情况。在维甲酸受体基因表达导致ALP水平升高的单层生长结束和开始多层生长与钙沉积之间，已被证明是MV释放的关键时间。因此，建立了钙化MV的采样标准。(3)用于快速磷酸酶检测的自动微量停流仪：在分级或动态测定的情况下，所构建的装置能够处理非常有限的MV制备量。利用上述新装置，从成骨细胞培养的MV被证明是一个卓有成效的实验系统。

The molecular process leading to apatite formation in matrix vesicle(MV) during the initial phase of calcification has never been understood so far on the basis of functional behavior of the intravesicular phosphatases. The project has been promoted with two types of MV participating each in different modes of calcification.(1) MV from enchondral ossification process: The MV from calf epiphyseal cartilage fractionated finally through Percoll density gradient centrifugation showed the highest alkaline phosphatase(ALP) specific activity among other organelles. The phosphotransferase activity of the ALP is interpreted as an essential function in calcification in or by MV. Apparent heterogeneity of MV suggested from the unfixed ratio of acid phosphatase(ACP)/ALP activity did not permit to ascribe the ACP as a unique vesicular enzyme despite possible localization of non-lyzosomal ACP in MV.(2) MV from intramembranous ossification process: The cultured cell of osteoblastic clone MC3T3-EI from mouse calvaria was examined in respect of the MV formation. In-between the ending of the monolayer growth where ALP level elevates in consequence of the retinoic acid receptor genes expression and the starting of the multi-layer growth together with Ca deposit, has been shown to be critical timing for the MV release. Thus, the criteria for sampling of calcifying MV have been established.(3) Automated micro stopped flow apparatus for rapid phosphatase assay: The apparatus constructed was powerful in dealing with very limited amounts of MV preparation in case of the fractionation or kinetic assay. Taking advantage of the above new apparatus, the MV from the osteoblastlike cultured cells is proved to be fruitful experimental system.