CALCIUM INDEPENDENT PLA2 AND ISCHEMIC CELL INJURY

非钙依赖性磷脂酶A2（CalPLA 2）在脑内的激活 近端小管通过形成 导致NaK-ATP酶严重抑制的有毒两亲物 在局部缺血期间。我们纯化了一种新的28 kDa的CaIPLA 2酶 从兔子的肾皮质中提取免疫组织化学研究使用抗- CaIPLA 2多克隆抗体已将该蛋白定位于 近端小管使用源自部分DNA的寡核苷酸探针， 从这种新的蛋白质获得的氨基酸序列，我们分离 兔肾皮质全长cDNA。该cDNA（CaIPLA 2-I） 编码一种分子量与28 kDa 从肾皮质纯化的蛋白质及其推导的氨基酸 序列包含三个肽的相同氨基酸序列 从纯化的28 kDa CaIPLA 2中分离。此外，它还 包含序列U-F-S-Q-G，与共识一致 序列周围的活性位点丝氨酸，G-X-S-X-U，观察到在 许多羧基酯酶包括脂肪酶。我们的假设是 近端小管中CaIPLA 2的激活有助于 在缺氧/缺血性细胞损伤期间显著地导致细胞死亡。 该项目旨在确定细胞和生物化学 CaIPLA 2激活导致肾上皮细胞 利用分子遗传学数据， 在初步研究中。我们建议：克隆并 表征CaIPLA 2基因在肾脏中表达。我们将 完成第二个部分cDNA克隆CalPLA的表征 2-2从兔肾和人肾中分离的CalPLA 2 cDNA。 二.为了确定磷酸化和钙在细胞凋亡中的作用， CaIPLA 2活性的调节。在大肠杆菌中表达CAIPLA 2蛋白， 昆虫细胞，并确定体外磷酸化的影响 CaIPLA 2活性。我们将研究在体内磷酸化的 CaIPLA 2在肾小管上皮细胞中的表达，我们还将 确定钙在膜易位中的作用和/或 CaIPLA 2的活化。特定氨基酸的磷酸化 残基将与酶活性相关。三.到 建立CaIPLA 2在缺血诱导的肾上皮细胞损伤中的作用 细胞损伤CaIPLA 2和肾单位的细胞分布 将建立体内缺血期间的区室化 以及连接CaIPLA 2和细胞激活的细胞事件 将定义缺血性细胞损伤期间的死亡。总的来说，这些 研究应有助于确定CaIPLA 2 激活导致局部缺血期间近端小管细胞死亡。

Activation of calcium-independent phospholipase A2 (CalPLA2) in the proximal tubule contributes to cell injury by the formation of toxic amphiphiles which cause profound inhibition of NaK-ATPase during ischemia. We have purified a novel 28 kDa CaIPLA 2 enzyme from rabbit kidney cortex. lmmunohistochemical studies using anti- CaIPLA 2 polyclonal antibodies have localized this protein to the proximal tubule. Using oligonucleotide probes derived from partial amino acid sequence obtained from this novel protein, we isolated a full length cDNA from rabbit kidney cortex. This cDNA (CaIPLA2-I) encodes for a protein with a similar molecular weight to the 28 kDa protein purified from kidney cortex and its deduced amino acid sequence contains the same amino acid sequence of three peptides isolated from the purified 28 kDa CaIPLA2. Furthermore, it also contains the sequence U-F-S-Q-G, which agrees with the consensus sequence surrounding active site serines, G-X-S-X-U, observed in many carboxyl esterases including lipases. Our hypothesis is that activation of CaIPLA2 in the proximal tubule contributes significantly to cell death during hypoxia/ischemic cell injury. This project seeks to determine the cellular and biochemical mechanisms by which CaIPLA2 activation leads to renal epithelial cell injury during ischemia using the molecular genetic data we gathered in preliminary studies. We propose: I. To clone and characterize CaIPLA2 genes expressed in the kidney. We will complete the characterization of a second partial cDNA clone CalPLA 2-2 isolated from rabbit kidney and a human kidney CalPLA2 cDNA. II. To determine the role of phosphorylation and calcium in the regulation of CaIPLA 2 activity. To express CAIPLA 2 protein in insect cells and determine the effects of in vitro phosphorylation on CaIPLA 2 activity. We will examine in vivo phosphorylation of CaIPLA2 in renal tubular epithelial cells, and also we will determine the role of calcium in membrane translocation and/or activation of CaIPLA 2. Phosphorylation at specific amino acid residues will be correlated with enzymatic activity. III. To establish the role of CaIPLA 2 in ischemia-induced renal epithelial cell injury. The cellular distribution of CaIPLA 2 and nephron compartmentalization during in vivo ischemia will be established and the cellular events that link activation of CaIPLA 2 and cell death during ischemic cell injury will be defined. Overall, these studies should help define the pathway(s) by which CaIPLA 2 activation leads to proximal tubule cell death during ischemia.