CALCIUM INDEPENDENT PLA2 AND ISCHEMIC CELL INJURY

钙非依赖性磷脂酶A2(CalPLA2)的激活 近端小管通过形成 对NAK-ATPase有明显抑制作用的有毒两亲化合物 在缺血期间。我们纯化了一种新的28 kDa CaIPLA2酶 取自兔肾皮质。免疫组织化学研究使用抗- CaIPLA2多克隆抗体已将该蛋白定位于 近端小管。使用来自部分基因的寡核苷酸探针 从这个新的蛋白质中获得了氨基酸序列，我们分离到了 兔肾皮质全长基因的克隆。该基因(CaIPLA2-I) 编码一种与28 kDa分子量相似的蛋白质 肾皮质蛋白及其推导的氨基酸的纯化 序列包含三个肽的相同氨基酸序列 从纯化的28 kDa CaIPLA2中分离得到。此外，它还 包含U-F-S-Q-G序列，与共识一致 活性中心丝氨酸G-X-S-X-U序列的观察 包括脂肪酶在内的许多羧基酯酶。我们的假设是 近端小管中CaIPLA2的激活起作用 显著促进缺氧/缺血细胞损伤过程中的细胞死亡。 这个项目试图确定细胞和生化 CaIPLA2激活导致肾上皮细胞损伤的机制 应用分子遗传学数据研究脑缺血时的细胞损伤 在初步研究中收集的。我们建议：一、克隆和 肾脏中表达的CaIPLA2基因的特征。我们会 完成第二个部分cDNA克隆Calpla的鉴定 从兔肾和人肾CalPLA2基因中分离得到2-2。 II.确定磷酸化和钙在细胞周期中的作用 CaIPLA2活性的调节。CAIPLA2蛋白在大肠杆菌中的表达 昆虫细胞和测定体外磷酸化的效果 对CaIPLA2活性的影响。我们将检测体内的磷酸化。 CaIPLA2在肾小管上皮细胞中的表达，我们也将 确定钙在膜转运和/或中的作用 CaIPLA2.特定氨基酸的磷酸化 残留物将与酶活性相关。三、至 CaIPLA2在缺血诱导的肾上皮细胞中的作用 细胞损伤。CaIPLA2和肾单位的细胞分布 体内缺血期间的分区将被建立 以及连接CaIPLA2激活和细胞的细胞事件 将对缺血细胞损伤期间的死亡进行定义。总的来说，这些 研究应有助于确定CaIPLA2通过的途径(S) 缺血时激活可导致近端小管细胞死亡。

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.