32Pi- and 45Ca-metabolism by matrix vesicle-enriched microsomes prepared from chicken epiphyseal cartilage by isosmotic Percoll density-gradient fractionation.
32Pi- and 45Ca-metabolism by matrix vesicle-enriched microsomes prepared from chicken epiphyseal cartilage by isosmotic Percoll density-gradient fractionation.
复制标题
通过等渗 Percoll 密度梯度分级从鸡骨骺软骨制备的富含基质囊泡的微粒体的 32Pi- 和 45Ca- 代谢。
DOI：
10.1007/bf02405054
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发表时间：
1983
影响因子：
4.2
通讯作者：
Wuthier,RE
中科院分区：
文献类型：
--
作者：
Warner,GP;Hubbard,HL;Lloyd,GC;Wuthier,RE
基质囊泡富集馏分分离从不同区域的骺软骨的非酶的方法，包括组织匀浆，差速离心，和等渗Percoll梯度分离。在20分钟至24小时的时间内同时研究了32 Pi和45 Ca的摄取。Percoll密度梯度分离骨骺微粒体成两个碱性磷酸酶丰富的部分：低密度noncalcifiable部分（P-I），和高密度的部分（P-II），容易矿化。P-II组分仅在生长板的钙化区域中发现。基于化学和物理性质和酶活性，这两个馏分是相似的，除了P-II含有显着更高水平的矿物离子比P-I，并有较低水平的碱性磷酸酶。这种矿物似乎主要以非晶体形式存在。P-II对~（32）Pi和~（45）Ca的代谢遵循一个复杂的动力学模式，在此模式中，这两种离子的大量积累之前是一个有限的初始摄取爆发和一个持续时间可变的滞后期。在矿物离子加载过程中，P-II组分的密度逐渐增加，这可以通过45 Ca、32 Pi和碱性磷酸酶的共迁移来证明。在早期矿物沉积期间（1-5小时），钙/磷吸收比非常低（1.0-1.2）和X-射线衍射图谱显示主要是无定形图案。这表明，在基质囊泡中积累的矿物质最初是某种形式的非结晶磷酸氢钙。L-四咪唑是一种有效的碱性磷酸酶抑制剂，在没有有机磷底物的情况下，抑制45 Ca和32 Pi的积累，32 Pi优先于45 Ca被抑制。这一发现，再加上最近的研究碱性磷酸酶在生理pH值的行为，表明该蛋白质不是作为磷酸水解酶，而是作为一个Pi结合或运输剂在囊泡介导的钙化。
Matrix vesicle-enriched fractions were isolated from different zones of epiphyseal cartilage by nonenzymatic methods involving tissue homogenization, differential centrifugation, and isosmotic Percoll gradient fractionation. Uptakes of both32Pi and45Ca were studied concomitantly over periods from 20 min to 24 h. Percoll density gradients separated epiphyseal microsomes into two alkaline phosphatase-rich fractions: a low-density noncalcifiable fraction (P-I), and a higher-density fraction (P-II) which readily mineralized. The P-II fraction was found only in calcifying regions of the growth plate. Based on chemical and physical properties and enzyme activities, both fractions were similar except that P-II contained significantly higher levels of mineral ions than did P-I, and had lower levels of alkaline phosphatase. The mineral appeared to be primarily in a noncrystalline form. Metabolism of32Pi and45Ca by P-II followed a complex kinetic pattern in which accumulation of large amounts of both ions was preceded by an initial limited burst of uptake and a lag-phase of variable duration. During mineral ion loading, the density of the P-II fraction progressively increased as evidenced by co-migration of45Ca,32Pi, and alkaline phosphatase to increasingly higher densities. During the period of early mineral deposition (1–5 h), Ca/P uptake ratios were very low (1.0–1.2) and X-ray diffraction patterns showed a predominantly amorphous pattern. This suggests that the mineral accumulated in matrix vesicles is initially some form of noncrystalline calcium monohydrogenphosphate. L-tetramisole, a potent inhibitor of alkaline phosphatase, inhibited accumulation of both45Ca and32Piin the absence of organic P substrates,32Pi being preferentially inhibited over45Ca. This finding, coupled with recent studies on the behavior of alkaline phosphatase at physiological pH, suggests that the protein is not acting as a phosphohydrolase, but rather as a Pi-binding or transport agent in vesicle-mediated calcification.