Biochemical Studies of 14 kDa Phospholipases A2

14 kDa 磷脂酶 A2 的生化研究

• 批准号: 6703048
• 负责人: Michael H Gelb
• 金额: $ 33.41万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-11 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6703048
• 关键词: X ray crystallography; active sites; caveolins; cell line; cell membrane; clinical research; electron microscopy; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; human tissue; immunocytochemistry; in situ hybridization; inflammation; membrane lipids; phospholipase A2; phospholipase C; phospholipase inhibitor; polymerase chain reaction; protein binding; protein structure function; receptor binding

DESCRIPTION: (provided by applicant) Phospholipases A2 (PLA2s) catalyze the hydrolysis of the sn-2 ester of membrane phospholipids to produce a free fatty acid and a lysophospholipid. There has been considerable interest in these enzymes because of their role in the liberation of arachidonic acid from mammalian cell membranes for the biosynthesis of the eicosanoid mediators of inflammation (prostaglandins, leukotrienes, and others). There is also considerable interest in these enzymes as a paradigm for understanding interfacial enzymology (the action of enzymes at the lipid-water interface). Many enzymes in cells operate on membranes. Mammalian cells contain two types of PLA2s that are involved in arachidonic acid production. The secreted PLA2s (sPLA2s) are 14-18 kDa, calcium-dependent enzymes that are secreted from a large number of mammalian cells following stimulation with pro-inflammatory mediators. Mammalian cells also contain an 87-kDA cytosolic PLA2 (cPLA2) that translocates to the membrane in response to a rise in cytosolic calcium. Recently we have cloned several new mouse and human sPLA2s. Thus, it is now clear that the sPLA2s constitute a superfamily of proteins in mammals. We have made significant progress toward the production of recombinant forms of the full set of mouse and human sPLA2s. We will complete this process and also study the interfacial kinetic and binding properties of these enzymes. We are now faced with the task of determining the expression profile of these sPLA2s in mammalian cells, and our studies will focus on cells from the airways of normal and asthmatic patients. It is well established that eicosanoids play a prominent role in causing many of the symptoms associated with asthma. It is important to determine which PLA2s are involved in releasing arachidonic acid. Such information will be useful in designing a new generation of therapeutics for treatment of airway inflammatory disorders. Once we have identified the PLA2s that are expressed in human airway cells, we will use PLA2-specific antibodies and small molecular weight inhibitors to probe whether these enzymes are involved in arachidonic acid release. The mechanisms by which PLA2s act in mammalian cells to liberate arachidonic acid are partially understood. Some sPLA2s act extracellularly on the plasma membrane of cells, while others are internalized into punctate intracellular membrane compartments. For the latter case, it is important to define this membrane compartment in more detail and to determine where the enzyme acts to release arachidonic acid. These studies will contribute to our fundamental understanding of how mammalian cells initiate the eicosanoid cascade.

描述：（由申请人提供）磷脂酶A2（PLA2S）催化 膜磷脂的Sn-2酯的水解以产生游离脂肪 酸和溶物磷脂。这些对这些引起了极大的兴趣 酶是因为它们在蛛网膜中的作用 哺乳动物细胞膜，用于生物合成的eicosanoid介质的生物合成 炎症（前列腺素，白细胞等）。也有 对这些酶的极大兴趣作为理解的范式 界面酶学（酶在脂质 - 水界面的作用）。 细胞中的许多酶在膜上作战。哺乳动物细胞包含两种类型 参与花生四烯酸产生的PLA2。分泌的pla2s （SPLA2S）是14-18 kDa，钙依赖性酶，这些酶是从A分泌的 促炎性刺激后，大量的哺乳动物细胞 调解人。哺乳动物细胞还包含87 kDa胞质PLA2（CPLA2） 响应胞质钙的升高而易位到膜。 最近，我们克隆了几只新的鼠标和人类SPLA2。因此，现在是 很明显，SPLA2S构成了哺乳动物中蛋白质的超家族。我们有 在生产重组形式的重组形式方面取得了重大进展 完整的鼠标和人类Spla2s。我们将完成此过程，也将 研究这些酶的界面动力学和结合特性。 我们现在面临确定这些表达式概况的任务 哺乳动物细胞中的SPLA2S，我们的研究将集中在气道的细胞上 正常和哮喘患者的众所周知，eicosanoids玩耍 引起与哮喘相关的许多症状的重要作用。这是 重要的是确定哪些PLA2涉及释放蛛网膜酸。 此类信息将在设计新一代治疗学方面很有用 用于炎症性疾病的治疗。一旦我们确定了 在人类气道细胞中表达的PLA2，我们将使用PLA2特异性 抗体和小分子量抑制剂可探测这些酶是否是否 参与花生四烯酸释放。 PLA2S在哺乳动物细胞中作用的机制释放蛛网膜菌 酸部分理解。一些SPLA2在血浆上细胞外作用 细胞的膜，而其他细胞被内在的细胞内内部 膜室。对于后一种情况，定义此问题很重要 膜室更详细，并确定酶的作用在哪里 释放花生四烯酸。这些研究将有助于我们的基本 了解哺乳动物细胞如何启动eicosanoid级联反应。