FUNCTION OF PURPLE ACID PHOSPHATASE IN DEVELOPING BONE

我们研究紫色、抗酒石酸的总体目标是 破骨细胞中的酸性磷酸酶(E_2)被用来描述其功能 并确定它是否起到了保护细胞的作用，以及 随之而来的骨重建，对抗铁中毒和 自由基。这项建议涵盖了体外培养的初始步骤 一项旨在确定E2：(1)是否可以在体内或 在细胞外，(2)受到与溶酶体不同的控制 水解酶，以及(3)清除铁和正磷酸盐 (正磷酸盐)。我们已经确定幼鼠的长骨 含有比酒石酸盐敏感(E1)高得多的E2水平 活性；我们还纯化了E2和免疫细胞化学 定位于破骨细胞囊泡。E2是唯一已知的酸 含有金属(即铁)部分的磷酸酶。单铁E2可能 其功能类似于转铁蛋白作为游离铁的水槽。两个铁杆 E2可能以两种方式发挥作用：(1)细胞内氧化还原电位 第二铁可以催化较低电位的氧化还原反应，并且 (2)如果第二个铁被还原，那么E2作为一种铁变得活跃 磷酸酶。但是，后两个可能的函数将 不会发生在吸收陷窝中的高正磷酸盐 内容物将保持E2的催化不活性。因此， 骨骼E2可能起到吸收游离铁的作用，这些游离铁将被保存下来 被结合在铁上的正磷酸盐固定。游离铁 在骨吸收过程中释放的物质必须被隔离，因为 腔隙的酸性和好氧环境有利于细菌的形成。 自由基。从分离的破骨细胞分泌E2，以及 外源铁和正磷酸盐的影响将是 用反向溶血空斑试验检测，它可以 显示单个细胞的分泌物。这些对分泌的影响将 也可以用免疫电泳法和荧光法进行定量 比标准分析灵敏1000倍的酶分析 对于雌二醇。我们将比较细胞内外的雌二醇水平 对于已知的两种溶酶体水解酶，E1和2- 葡萄糖醛酸苷酶，以确定可能的行为差异 在酶之间。后两种酶的检测将使用 与E2相同的荧光检测方法，并将用于 区分E1和E2。如果结果支持分泌 E2尤其是对外源铁和正磷酸盐的反应，它 将确定E2是否能清除这些离子。激活 添加第二个铁原子的E_2磷酸酶活性 E2及其随后与抗坏血酸的减量将作为 铁清除试验的基础；E2的失活 正磷酸盐将形成清除作用的化验基础 正磷酸盐。

The overall goals of our research on purple, tartrate-resistant acid phosphatase (E2) in osteoclasts are to delineate its function and determine if it serves a role in protecting the cell, and subsequent bone remodeling, against damage by iron toxicity and free radicals. This proposal covers initial steps of an in vitro study designed to determine if E2: (1) can function inside or outside the cell, (2) is under different controls than are lysosomal hydrolases, and (3) scavenges iron and orthophosphate (orthophosphate). We have determined that long bones of rat pups contain much higher levels of E2 than of tartrate-sensitive (E1) activity; we also have purified E2 and immunocytochemically localized it to osteoclast vesicles. E2 is the only known acid phosphatase with a metal (i.e. iron) moiety. One-iron E2 may function similarly to transferrin as a sink for free iron. Two-iron E2 may function in two ways: (1) The redox potential of the second iron can catalyze redox reactions of lower potential, and (2) if the second iron is reduced, then E2 becomes active as a phosphatase. However, the latter two possible functions would not occur in resorption lacunae where the high orthophosphate content would maintain E2 catalytically inactive. Therefore, bone E2 may function as a sink for free iron, which would be kept immobilized by orthophosphate bound to the iron. Free iron released during bone resorption must be sequestered because the acidic and aerobic milieu of the lacunae would favor formation of free radicals. The secretion of E2 from isolated osteoclasts, and the effects of exogenous iron and orthophosphate will be determined with a reverse hemolytic plaque assay, which can show secretion by single cells. These effects on secretion will also be quantitated with immunoelectrophoresis and a fluorescent enzyme assay that is 1000x more sensitive than standard assays for E2. Levels of E2 inside and outside the cell will be compared to those of two known lysosomal hydrolases, E1 and 2- glucuronidase, to determine possible differences in behavior between the enzymes. Assays for the latter two enzymes will use the same fluorescent assay as for E2, and means will be used to distinguish between E1 and E2. If the results support secretion of E2 especially in response to exogenous iron and orthophosphate, it will be determined if E2 can scavenge these ions. Activation of E2 phosphatase activity by the addition of the second iron atom to E2, and its subsequent reduction with ascorbate, will serve as the basis of an assay for iron scavenging; inactivation of E2 with orthophosphate will form the basis of an assay for the scavenging of orthophosphate.