MATRIX VESICLES AND CALCIFICATION

本研究的长远目标是阐明其发病机制。 软骨内钙化，这是正常骨骼必不可少的过程 形成、骨骼发育和骨折愈合。而其他人 有多种因素参与，基质小泡(MV)主要参与 开始钙化。因此，本研究的目的是阐明 MV钙化。MV，当从生长板软骨和 在合成软骨淋巴中孵化，通过以下方式诱导矿物质形成 获得大量的钙离子和磷酸盐。MV中含有高水平的 矿化作用。这个项目的三个主要目标是：1) 鉴定关键的MV蛋白，2)鉴定病毒核型 复合体；3)重建功能性MV。拳头，离子搬运工 Ca~(2+)和P~(2+)进入囊腔是必需的 将对MV矿化进行表征。钙离子转运蛋白，膜联蛋白V， 还具有胶原蛋白结合活性。由于与类型交互 II型和X型胶原激活钙离子进入MV，假设 胶原蛋白结合激活膜联蛋白的钙通道将被检测。 MV中的PI转运蛋白知之甚少；利用大鼠肾脏的PI转运蛋白 以c DNA为探针，鉴定和克隆软骨细胞PI转运蛋白 以帮助鉴定其在MV PI转运中的活性。入口处 MV矿化过程中Ca~(2+)进入MV激活磷脂酶 选择性地分解磷脂酰丝氨酸和鞘磷脂。因为 这些脂类阻碍了来自囊泡管腔的矿物质的生长 磷脂酶将被分离和鉴定。另外，一种酸不稳定的 核复合体和钙/磷结合蛋白被发现是 对MV矿物形成的诱导至关重要。因此，这 核复合体(电解质、脂类和蛋白质)，以及 将分离囊泡管腔内的钙/磷结合蛋白，并 特色化的。最后，确定了这些基本组件后， 核复合体和功能MV将通过以下方式重组 将这些关键的蛋白质、矿物质离子和脂类合成成 单层囊泡。1)Annexin V和Annexin V的功能 对其钙通道活性的调节将继续探索；2) MV依赖Na+的PI转运体，3)MV核心蛋白参与 储存Ca~(2+)和PI，4)负责分解的磷脂酶 MV膜在钙化过程中，以及5)核复合体将 与世隔绝，独树一帜。最后，有了这些信息， 成核复合体，以及完整的功能MV，将是 使用脂类、电解质、蛋白质和酶进行重组 成为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.