FUNCTION OF PURPLE ACID PHOSPHATASE IN DEVELOPING BONE

我们研究紫色、抗酒石酸盐 破骨细胞中酸性磷酸酶（E2）的作用 并确定它是否在保护细胞中起作用， 随后的骨重建，对抗铁毒性造成的损伤， 自由基 本提案涵盖了体外 研究旨在确定E2：（1）是否可以在体内发挥作用， 在细胞外，（2）受到与溶酶体不同的控制 水解酶，和（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;我们也有纯化的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 磷酸酶与金属（即铁）部分。 单铁E2可 其功能类似于转铁蛋白作为游离铁的接收器。 二号铁杆 E2可以以两种方式起作用：（1）E2的氧化还原电位可以与E2的氧化还原电位相同。 第二种铁可以催化较低电位的氧化还原反应，和 (2)如果第二个铁被还原，那么E2就变成活性的铁。 磷酸酶。 然而，后两种可能的功能将 不发生在吸收陷窝，其中高正磷酸盐 含量将保持E2催化失活。 因此，我们认为， 骨E2可以作为游离铁的汇， 被与铁结合的正磷酸盐固定。 游离铁 在骨吸收过程中释放的，必须被隔离， 腔隙的酸性和有氧环境将有利于形成 自由基 从分离的破骨细胞分泌E2， 外源性铁和正磷酸盐的影响将是 用反向溶血空斑试验测定， 显示单细胞分泌。 这些对分泌的影响将 也可以用免疫电泳和荧光定量 比标准检测灵敏1000倍的酶检测 对于E2。 将比较细胞内外的E2水平 与两种已知的溶酶体水解酶E1和2-相比， 葡萄糖醛酸酶，以确定可能的行为差异 在酶之间。 后两种酶的测定将使用 与E2相同的荧光测定，将使用方法 区分E1和E2。 如果结果支持分泌 E2特别是对外源性铁和正磷酸盐的反应，它 将确定E2是否能吸收这些离子。 激活 E2磷酸酶活性通过添加第二个铁原子到 E2及其随后与抗坏血酸的还原作用将作为 铁清除试验的基础; E2的灭活 正磷酸盐将形成用于清除 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.