Biochemical Studies of 14 kDa Phospholipases A2

14 kDa 磷脂酶 A2 的生化研究

基本信息

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
项目类别：
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

项目摘要

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 (PLA2) 催化膜磷脂的 sn-2 酯水解产生游离脂肪酸和溶血磷脂。人们对这些内容产生了浓厚的兴趣酶，因为它们在从花生四烯酸中释放花生四烯酸方面发挥作用哺乳动物细胞膜用于生物合成类二十烷酸介质炎症（前列腺素、白三烯等）。还有人们对这些酶非常感兴趣，将其作为理解的范例界面酶学（酶在脂质-水界面上的作用）。细胞中的许多酶在膜上发挥作用。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 的钙依赖性酶，由促炎刺激后产生大量哺乳动物细胞调解员。哺乳动物细胞还含有 87 kDA 胞质 PLA2 (cPLA2)，响应细胞质钙的增加而易位至膜。最近我们克隆了几个新的小鼠和人类 sPLA2。因此，现在是明确 sPLA2 构成哺乳动物中的蛋白质超家族。我们有在重组形式的生产方面取得了重大进展全套小鼠和人类 sPLA2。我们将完成这个过程，并且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，我们的研究将集中于气道细胞正常人和哮喘患者。众所周知，类二十烷酸具有在引起许多与哮喘相关的症状中起重要作用。这是确定哪些 PLA2 参与释放花生四烯酸很重要。这些信息将有助于设计新一代疗法用于治疗气道炎症性疾病。一旦我们确定了 PLA2 在人类气道细胞中表达，我们将使用 PLA2 特异性抗体和小分子量抑制剂来探测这些酶是否参与花生四烯酸的释放。 PLA2 在哺乳动物细胞中作用以释放花生四烯酸的机制酸已被部分了解。一些 sPLA2 在细胞外作用于血浆细胞膜，而其他则内化到点状细胞内膜隔室。对于后一种情况，定义这一点很重要更详细地了解膜室并确定酶作用的位置释放花生四烯酸。这些研究将有助于我们的基础研究了解哺乳动物细胞如何启动类二十烷酸级联。