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：(申请人提供)磷脂酶A2(PLA2)催化 膜磷脂sn-2酯的水解生成游离脂肪 酸和一种溶血磷脂。人们对这些已经很感兴趣了 酶，因为它们在花生四烯酸从 用于生物合成二十烷类介体的哺乳动物细胞膜 炎症(前列腺素、白三烯等)。还有就是 对这些酶作为理解范例的相当大的兴趣 界面酶学(酶在脂-水界面的作用)。 细胞中的许多酶在膜上工作。哺乳动物细胞包含两种类型 参与花生四烯酸生产的PLA2。分泌的PLA2 (SPLA2)是14-18 kDa的钙依赖酶，由一种 促炎因子刺激后的大量哺乳动物细胞 调解人。哺乳动物细胞也含有一个87kDA的胞浆磷脂酶A2(CPLA2)，它 细胞内钙离子升高，转位到细胞膜上。 最近，我们克隆了几个新的小鼠和人的sPLA2。因此，现在是 很明显，sPLA2构成了哺乳动物中的一个蛋白质超家族。我们有 在重组形式的生产方面取得了重大进展 全套老鼠和人类的sPLA2。我们将完成这一进程，并 研究这些酶的界面动力学和结合特性。 我们现在面临的任务是确定这些基因的表达谱 哺乳动物细胞中的sPLA2，我们的研究将集中在来自呼吸道的细胞 正常患者和哮喘患者。众所周知，二十烷类化合物扮演着 在引起许多与哮喘相关的症状方面起着突出的作用。它是 重要的是确定哪些PLA2参与释放花生四烯酸。 这些信息将有助于设计新一代疗法 用于治疗呼吸道炎症性疾病。一旦我们确定了 在人类呼吸道细胞中表达的PLA2，我们将使用PLA2特异性的 抗体和小分子量抑制剂来检测这些酶 都参与花生四烯酸的释放。 PLA2在哺乳动物细胞中释放花生四烯酸的机制 人们对酸有一定的了解。一些sPLA2在细胞外作用于血浆。 细胞膜，而其他的则内化成点状的细胞内 隔膜隔间。对于后一种情况，重要的是定义这一点 更详细的细胞膜隔间，并确定酶在哪里作用 释放花生四烯酸。这些研究将有助于我们从根本上 了解哺乳动物细胞如何启动二十烷基磺酸盐的级联反应。