MATRIX VESICLES AND CALCIFICATION

本研究的长远目标是阐明 软骨内钙化，正常骨骼的一个基本过程 形成、骨骼发育和骨折愈合。 而其他 因子参与，基质囊泡（MV）主要涉及 开始钙化。 因此，本研究的目的是阐明 二尖瓣钙化。 MV，当从生长板软骨分离时， 在人造软骨淋巴液中孵育，诱导矿物质形成， 获得大量的Ca 2+和Pi。 MV含有高水平的 成矿 该项目的三个主要目标是：1） 表征关键MV蛋白，2）表征核结构蛋白， 复合物，和3）重建功能MV。 拳头，离子搬运工 对Ca ~（2+）和Pi进入囊泡腔至关重要， 将对MV矿化进行表征。 钙转运蛋白，膜联蛋白V， 还具有胶原结合活性。 由于与类型的交互 II和X胶原激活Ca 2+进入MV，这一假设是， 将测试胶原结合激活膜联蛋白Ca 2+通道。 MV中的Pi转运蛋白知之甚少;使用大鼠肾脏Pi转运蛋白 cDNA作为探针，鉴定和克隆软骨细胞Pi-转运蛋白 以帮助表征其在MV Pi转运中的活性。 门口 在MV矿化过程中进入MV的Ca 2+激活磷脂酶， 选择性分解磷脂酰丝氨酸和鞘磷脂。 因为 这些脂质阻碍了矿物质从囊泡腔中生长出来， 磷脂酶将被分离和表征。 此外，酸不稳定 核复合物和Ca ~（2+）/Pi结合蛋白被发现是 对诱导MV矿物形成至关重要。 因此，这 核复合物（电解质，脂质和蛋白质），以及 将分离囊泡腔中的Ca 2 +/Pi结合蛋白， 表征了 最后，确定了这些基本组件后， 核复合物和功能性MV将通过 将这些关键的蛋白质、矿物质离子和脂质结合到合成的 单层囊泡。 总结如下：1）膜联蛋白V和膜联蛋白V的功能 将继续探索其Ca 2+通道活性的调节; 2） MV Na+依赖的Pi-转运蛋白，3）MV核心蛋白参与 储存Ca 2+和Pi，4）负责分解 MV膜在钙化过程中，和5）核复合体将 被隔离和特征化。 最后，有了这些信息，（6） 核复合物，和7）完整的功能MV，将是 使用脂质、电解质、蛋白质和酶进行重构， 是MV功能的关键。

The long-range of this research is to elucidate the mechanism of endochondral calcification, a process essential for normal bone formation, skeletal development and fracture healing. While other factors are involved, matrix vesicles (MV) are primarily implicated in initiating calcification. Thus the goal of this research is to elucidate MV calcification. MV, when isolated from growth plate cartilage and incubated in a synthetic cartilage lymph, induces mineral formation by acquiring large amounts of Ca2+ and Pi. MV contain high levels of mineralization. The three major goals of this project are: 1) to characterize key MV proteins, 2) to characterize the nucleational complex, and 3) to reconstitute functional MV. Fist, ion porters essential for the entrance of Ca2+ and Pi into the vesicle lumen during MV mineralization will be characterized. The Ca2+ porter, annexin V, also possesses collagen-binding activities. Since interaction with type II and X collagens activates Ca2+ entrance into MV, the hypothesis that collagen binding activates the annexin Ca2+ channel will be tested. Little is known of the Pi-porter in MV; using rat kidney Pi-transporter cDNA as a probe, the chondrocyte Pi-porter will be identified and cloned to aid in characterizing its activity in MV Pi-transport. Entrance of Ca2+ into MV during MV mineralization activates phospholipases that selectively break down phosphatidylserine and sphingomyelin. Because these lipids impede outgrowth of mineral from the vesicle lumen, MV phospholipases will be isolated and characterized. Also, an acid-labile nucleational complex and Ca2+/Pi-binding proteins have been found to be critical for induction of MV mineral formation. Accordingly, this nucleational complex (electrolytes, lipids and proteins), and the Ca2+/Pi-bindings proteins in the vesicle lumen, will be isolated and characterized. Finally, with these essential components identified, the nucleational complex, and functional MV, will be reconstituted by incorporating these key proteins, mineral ions and lipids into synthetic unilamellar vesicles. To summarize: 1) the functions of annexin V and regulation of its Ca2+ channel activity will continue to be explored; 2) the MV Na+-dependent Pi-transporter, 3) the MV core proteins involved in storing Ca2+ and Pi, 4) phospholipases responsible for breakdown of the MV membrane during calcification, and 5) the nucleational complex will be isolated and characterized. Finally, with this information, 6) the nucleational complex, and 7) complete functional MV, will be reconstituted using lipids, electrolytes, proteins and enzymes shown to be key to MV function.