Wu L N, Genge B R, Dunkelberger D G, LeGeros R Z, Concannon B, Wuthier R E
Laboratory for Biomineralization Research, Department of Chemistry and Biochemistry, and School of Medicine, University of South Carolina, Columbia, South Carolina 29208, USA.
J Biol Chem. 1997 Feb 14;272(7):4404-11. doi: 10.1074/jbc.272.7.4404.
While previous studies revealed that matrix vesicles (MV) contain a nucleational core (NC) that converts to apatite when incubated with synthetic cartilage lymph, the initial mineral phase present in MV is not well characterized. This study explored the physicochemical nature of this Ca2+ and Pi-rich NC. MV, isolated from growth plate cartilage, were analyzed directly by solid-state 31P NMR, or incubated with hydrazine or NaOCl to remove organic constituents. Other samples of MV were subjected to sequential treatments with enzymes, salt solutions, and detergents to expose the NC. We examined the NC using transmission electron microscopy, energy-dispersive analysis with x-rays, and electron and x-ray diffraction, Fourier transform-infrared spectroscopy, high performance thin-layer chromatographic analysis, and SDS-polyacrylamide gel electrophoresis. We found that most of the MV proteins and lipids could be removed without destroying the NC; however, NaOCl treatment annihilated its activity. SDS-polyacrylamide gel electrophoresis showed that annexin V, a phosphatidylserine (PS)-dependent Ca2+-binding protein, was the major protein in the NC; high performance thin-layer chromatographic analysis revealed that the detergents removed the majority of the polar lipids, but left significant free cholesterol and fatty acids, and small but critical amounts of PS. Transmission electron microscopy showed that the NC was composed of clusters of approximately 1.0 nm subunits, which energy-dispersive analysis with x-rays revealed contained Ca and Pi with a Ca/P ratio of 1.06 +/- 0. 01. Electron diffraction, x-ray diffraction, and Fourier transform-infrared analysis all indicated that the NC was noncrystalline. 1H-Cross-polarization 31P NMR indicated that the solid phase of MV was an HPO42--rich mixture of amorphous calcium phosphate and a complex of PS, Ca2+, and Pi. Taken together, our findings indicate that the NC of MV is composed of an acid-phosphate-rich amorphous calcium phosphate intermixed with PS-Ca2+-Pi, annexin V, and other proteins and lipids.
虽然先前的研究表明基质小泡（MV）含有一个成核核心（NC），当与合成软骨淋巴液一起孵育时会转化为磷灰石，但MV中存在的初始矿物相尚未得到很好的表征。本研究探讨了这种富含Ca2+和Pi的NC的物理化学性质。从生长板软骨中分离出的MV，直接通过固态31P NMR进行分析，或与肼或次氯酸钠孵育以去除有机成分。MV的其他样品依次用酶、盐溶液和去污剂处理以暴露NC。我们使用透射电子显微镜、能量色散X射线分析、电子和X射线衍射、傅里叶变换红外光谱、高效薄层色谱分析和SDS聚丙烯酰胺凝胶电泳来检查NC。我们发现，在不破坏NC的情况下，可以去除大部分MV蛋白和脂质；然而，次氯酸钠处理会消除其活性。SDS聚丙烯酰胺凝胶电泳显示，膜联蛋白V，一种依赖磷脂酰丝氨酸（PS）的Ca2+结合蛋白，是NC中的主要蛋白；高效薄层色谱分析表明，去污剂去除了大部分极性脂质，但留下了大量游离胆固醇和脂肪酸，以及少量但关键的PS。透射电子显微镜显示，NC由约1.0 nm亚基的簇组成，能量色散X射线分析表明其含有Ca和Pi，Ca/P比为1.06±0.01。电子衍射、X射线衍射和傅里叶变换红外分析均表明NC是非晶态的。1H交叉极化31P NMR表明，MV的固相是富含HPO42-的无定形磷酸钙与PS、Ca2+和Pi的复合物的混合物。综上所述，我们的研究结果表明，MV的NC由富含酸性磷酸盐的无定形磷酸钙与PS-Ca2+-Pi、膜联蛋白V以及其他蛋白质和脂质混合而成。
