HUMAN LIPOXYGENASES--PURIFICATION AND MOLCULAR PROBES

脂氧合酶是一类非血红素型含铁双加氧酶 它们已经被证明能产生广泛的生理上的 活性代谢物。12-脂氧合酶(12-Lx)存在于 血小板、白细胞、肺和脑。12-LX将花生四烯酸转化为 12-HPETE，然后可以转换为12-HETE。最近，环氧树脂 12的二羟基和三羟基衍生物(S)- HPETE已被描述。酶5-脂氧合酶(5-Lx)起着 在白三烯的生物合成中起关键作用，白三烯在 对许多生物过程的调节，从急性 炎症和血栓形成导致慢性心血管疾病，如 动脉硬化。15-脂氧合酶(15-Lx)会发生过氧化 花生四烯酸生成15(S)-HPETE，可转化为15(S)-HETE 和三羟基化合物脂氧素A(4)和脂氧素B(4)，它们表现出 一系列的生物活动。纯净品的商业化供应 人脂氧合酶的制备及一整套分子探针 (核酸探针以及多克隆抗体和单抗) 极大地促进了我们对生理学和 这些蛋白质的病理生理作用。 该项目的最终目标是开发具有商业可行性的 人脂肪氧合酶均一性的纯化方法 多克隆和单克隆抗体以及核酸的生产 针对每种酶的探针。然后这些试剂将被 销售给研究病毒分布和监管的科学家 人5-、12-和15-脂氧合酶在正常和疾病中的表达 个人。在第一阶段，我们建议进一步发展方法，以 商品用人血小板12-LX的稳定和纯化 生产，并将产生兔多克隆抗体特异性 对于这种蛋白质。在随后的第二阶段，我们计划：(A)产生 抗血小板12-LX的单抗，(B)纯化人5-LX， 15-LX和白细胞12-LX，并产生多克隆和单抗 针对这些酶中的每一个，(C)合成额外的核酸 探头，根据需要为人类提供一整套探头 脂氧合酶，和(D)筛选已建立的人类细胞系，以便 确定表达和活性检测的便利标准 这些酶中的每一种。

Lipoxygenases constitute a family of non-heme iron-containing dioxygenases that have been demonstrated to produce a wide range of physiologically active metabolites. The enzyme 12-lipoxygenase (12-LX) is present in platelets, leukocytes, lung and brain. 12-LX converts arachidonic acid to 12-HPETE, which can then be converted to 12-HETE. More recently, epoxy (hepoxylins A(3) and B(3)) dihydroxy and trihydroxy derivatives of 12(S)- HPETE have been described. The enzyme 5-lipoxygenase (5-LX) plays a pivotal role in the biosynthesis of leukotrienes, which are important in the regulation of numerous biological processes, ranging from acute inflammation and thrombosis to chronic cardiovascular disorders such as atherosclerosis. The enzyme 15-lipoxygenase (15-LX) peroxidizes arachidonic acid to form 15(S)-HPETE, which can be converted to 15(S)-HETE and the trihydroxy compounds lipoxin A(4) and lipoxin B(4), which exhibit a range of biological activities. Commercial availability of purified preparations of human lipoxygenases and a complete set of molecular probes (nucleic acid probes as well as poly- and monoclonal antibodies) will greatly facilitate our understanding of the physiologic and pathophysiologic roles of these proteins. The ultimate goal of this project is the development of commercially viable methods for purification to homogeneity of human lipoxygenases, the production of polyclonal and monoclonal antibodies, as well as nucleic acid probes specific for each of these enzymes. These reagents will then be marketed to scientists investigating the distribution and regulation of human 5-, 12- and 15-lipoxygenase enzymes in normal and diseased individuals. In Phase I, we propose to further develop methods to stabilize and purify human platelet 12-LX suitable for commercial production, and will also generate rabbit polyclonal antibodies specific for this protein. In subsequent Phase II, we plan to: (a) generate monoclonal antibodies specific for platelet 12-LX, (b) purify human 5-LX, 15-LX and leukocyte 12-LX and generate polyclonal and monoclonal antibodies specific for each of these enzymes, (c) synthesize additional nucleic acid probes, as needed, to provide a complete set of probes for human lipoxygenases, and (d) screen established human cell lines in order to identify convenience standards for assays of expression and activity of each of these enzymes.