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

在钙化初始阶段，基质囊泡（MV）中导致磷灰石形成的分子过程迄今尚未基于囊泡内磷酸酶的功能行为被理解。两种MV以不同的钙化模式参与了项目的推进。(1)内软骨成骨过程的碱性磷酸酶（ALP）比活性：经Percoll密度梯度离心分离最终分离的小牛骨骺软骨的碱性磷酸酶（ALP）比活性在其他细胞器中最高。ALP的磷酸转移酶活性被解释为MV中或由MV引起的钙化的重要功能。从酸性磷酸酶(ACP)/ALP活性的不固定比率来看，MV具有明显的异质性，尽管可能在MV中定位了非溶酶体ACP，但不能将ACP归因于独特的囊泡酶。(2)膜内骨化过程中的MV：用小鼠颅骨成骨细胞MC3T3-EI培养细胞，观察其MV的形成情况。由于维甲酸受体基因表达导致ALP水平升高，在单层生长结束和多层生长开始以及Ca沉积之间，已被证明是MV释放的关键时间。从而建立了钙化MV的采样标准。(3)用于快速磷酸酶测定的自动微停流装置：在分馏或动力学测定中，该装置在处理非常少量的MV制剂方面功能强大。利用上述装置，从成骨样培养细胞中提取MV是一种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.