Wu L N, Yoshimori T, Genge B R, Sauer G R, Kirsch T, Ishikawa Y, Wuthier R E
Department of Chemistry and Biochemistry, School of Medicine, University of South Carolina, Columbia 29208.
J Biol Chem. 1993 Nov 25;268(33):25084-94.
The factors that drive mineralization of matrix vesicles (MV) have proven difficult to elucidate; in the present studies, various detergent, chemical, and enzyme treatments were used to reveal the nature of the nucleational core. Incubation with detergents that permeabilized the membrane enhanced calcification of treated MV incubated in synthetic cartilage lymph. While detergents removed most of the membrane lipid, they left significant amounts of the MV annexins and nearly all of the Ca2+, Pi, and Zn2+. Extraction with 1 M NaCl removed much of the Ca2+ and Pi present in MV, markedly reducing Ca2+ accumulation; these effects could be prevented by low levels of Ca2+ and Pi in the NaCl extractant. Treatment with chymotrypsin appeared to damage proteins required for MV mineralization; further treatment with detergents to bypass the membrane reactivated MV mineralization. Treatment of MV with pH 6 citrate removed Ca2+ and Pi, destroying their ability to mineralize; subsequent treatment with detergents did not reactivate these MV. Incubation of the detergent-resistant core with o-phenanthroline complexed Zn2+ and stimulated mineralization; addition of Zn2+ to synthetic cartilage lymph blocked the ability of the core to mineralize. These studies show that once the nucleational core complex is formed, the membrane-enclosed domain is no longer essential for MV calcification. Our findings indicate that the MV core contains two main components as follows: a smaller membrane-associated complex of Ca2+, Pi, phosphatidylserine, and the annexins that nucleates crystalline mineral formation, and a larger pool of Ca2+ and Pi bound to lumenal proteins. These proteins appear to bind large amounts of mineral ions, stabilize the nucleational complex, and aid its transformation to the first crystalline phase. Once nucleated, the crystalline phase appears to feed on protein-bound mineral ions until external ions enter through the MV ion channels. Zn2+ appears to regulate gating of the ion channels and conversion of the nucleational complex to the crystalline state.
已证实，驱动基质小泡（MV）矿化的因素难以阐明；在本研究中，采用了各种去污剂、化学和酶处理方法来揭示成核核心的性质。用能使膜通透的去污剂孵育可增强在合成软骨淋巴液中孵育的经处理MV的钙化。虽然去污剂去除了大部分膜脂质，但它们留下了大量的MV膜联蛋白以及几乎所有的Ca2+、Pi和Zn2+。用1 M NaCl提取去除了MV中存在的大部分Ca2+和Pi，显著降低了Ca2+的积累；NaCl提取剂中低水平的Ca2+和Pi可防止这些影响。用胰凝乳蛋白酶处理似乎会破坏MV矿化所需的蛋白质；用去污剂进一步处理以绕过膜可重新激活MV矿化。用pH 6柠檬酸盐处理MV会去除Ca2+和Pi，破坏其矿化能力；随后用去污剂处理并不能重新激活这些MV。用邻菲罗啉络合Zn2+孵育抗去污剂核心并刺激矿化；向合成软骨淋巴液中添加Zn2+会阻止核心的矿化能力。这些研究表明，一旦形成成核核心复合物，膜封闭区域对于MV钙化就不再是必需的。我们的研究结果表明，MV核心包含两个主要成分：一个较小的与膜相关的Ca2+、Pi、磷脂酰丝氨酸和膜联蛋白复合物，它启动晶体矿物质形成；以及一个较大的与腔蛋白结合的Ca2+和Pi池。这些蛋白质似乎结合大量的矿质离子，稳定成核复合物，并帮助其转变为第一个晶相。一旦开始成核，晶相似乎依赖于与蛋白质结合的矿质离子，直到外部离子通过MV离子通道进入。Zn2+似乎调节离子通道的门控以及成核复合物向晶体状态的转变。
