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

迄今为止，在钙化的初始阶段导致基质囊泡（MV）中磷灰石形成的分子过程从未基于囊泡内磷酸酶的功能行为而被理解。该项目已经推广，两种类型的MV分别参与不同的钙化模式。(1)软骨内成骨过程中的细胞器：Percoll密度梯度离心分离的小牛骺软骨中的细胞器碱性磷酸酶（ALP）比活性最高。ALP的磷酸转移酶活性被解释为MV中或由MV引起的钙化的基本功能。从酸性磷酸酶（ACP）/ALP活性的不固定的比例建议MV的明显的异质性不允许归因于ACP作为一个独特的囊泡酶，尽管可能的本地化的非溶酶体ACP在MV。(2)膜内成骨过程中的MV：检测培养的小鼠颅骨成骨细胞克隆MC 3 T3-EI的MV形成。在单层生长的结束（其中ALP水平由于视黄酸受体基因表达而升高）和多层生长连同Ca存款的开始之间，已被证明是MV释放的关键时间。因此，钙化MV的采样标准已经建立。(3)用于快速磷酸酶测定的自动化微断流装置：在分级分离或动力学测定的情况下，所构建的装置在处理非常有限量的MV制备方面是强大的。利用上述新装置，从成骨样细胞培养的MV被证明是一个富有成效的实验系统。

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.