Action of Phospholipase A2 on Apoptotic Cells

描述（由申请人提供）：分泌磷脂酶A2 （sPLA2）与脂质双分子层结合并催化磷脂水解。正常情况下，细胞会抵抗这种酶的作用，但在细胞凋亡或外伤时，它们会变得易感。膜表面的水解显然需要两个步骤：酶吸附到膜表面，然后磷脂从正常的双分子层位置迁移到酶的活性位点。以红细胞为模型的实验表明，当细胞易受sPLA2影响时，有序脂质和无序脂质结构域之间的边界会增殖。假设相邻磷脂之间在这些边界处的有利相互作用的减少可以通过促进磷脂迁移到酶活性位点来增强sPLA2的活性。本研究将把这些研究扩展到有核细胞，并在激素刺激的细胞凋亡过程中验证这一假设。他们会问四个问题。1)在细胞凋亡过程中是否发生了膜序的改变，它们是否减少了磷脂与邻体的相互作用？2)细胞凋亡如何增加sPLA2的易感性；它是促进酶在膜表面的增强吸附，脂质迁移到被吸附酶的活性部位，还是两者兼而有之？3)假设在理论上可行吗？4)这些机制如何适用于不同类型的哺乳动物sPLA2？为了回答这些问题，我们将采用六种常规方法来研究地塞米松刺激淋巴瘤细胞凋亡过程中的变化。首先，膜物理性质的改变将通过荧光光谱和使用膜探针的显微镜检查。其次，磷脂-邻体相互作用的强度将通过merocyanine 540的荧光和测量白蛋白从细胞膜中提取荧光磷脂的速率来评估。第三，将在不同的酶浓度下测定膜水解动力学，并在上述两步模型的背景下进行数学分析。这些实验将重复使用各种形式的哺乳动物sPLA2。第四，该假设将通过计算机模拟进行理论评估。第五，将测量sPLA2与细胞膜表面的结合。第六，通过测定sPLA2对磷脂的提取率来评估磷脂迁移到结合酶活性位点的能力。

DESCRIPTION (provided by applicant): Secretory phospholipase A2 (sPLA2) binds to lipid bilayers and catalyzes hydrolysis of phospholipids. Normally, cells resist the enzyme's action, but they become susceptible during apoptosis or trauma. Hydrolysis at the membrane surface apparently requires two steps: enzyme adsorption to the membrane surface followed by migration of phospholipids from their normal bilayer position up into the enzyme's active site. Experiments using erythrocytes as a model suggest that when cells become susceptible to sPLA2, boundaries between domains of ordered and disordered lipids proliferate. Reduction of favorable interactions among neighboring phospholipids at those boundaries is hypothesized to enhance sPLA2 activity by facilitating migration of phospholipids into the enzyme active site. This proposal will extend these studies to nucleated cells and test the hypothesis during hormone-stimulated apoptosis. Four questions will be asked. 1) Do changes in membrane order occur during apoptosis and do they reduce phospholipids-neighbor interactions? 2) How does apoptosis increase susceptibility to sPLA2; does it promote enhanced adsorption of the enzyme to the membrane surface, migration of lipids into the active site of the adsorbed enzyme, or both? 3) Is the hypothesis theoretically feasible? 4) How do these mechanisms apply to the various types of mammalian sPLA2? To answer these questions, six general procedures will be used to study changes in lymphoma cells during apoptosis stimulated by dexamethasone. First, alterations to membrane physical properties will be examined by fluorescence spectroscopy and microscopy using the membrane probe laurdan. Second, the strength of phospholipid-neighbor interactions will be assessed by the fluorescence of merocyanine 540 and by measuring the rate at which albumin extracts fluorescent phospholipids from the cell membrane. Third, the kinetics of membrane hydrolysis will be assayed at various enzyme concentrations and mathematically analyzed in the context of the two-step model described above. These experiments will be repeated using various forms of mammalian sPLA2. Fourth, the hypothesis will be evaluated theoretically by computer simulations. Fifth, the binding of sPLA2 to the surface of the cell membranes will be measured. Sixth, the ability of phospholipids to migrate to the active site of bound enzyme will be assessed by measuring the rate of extraction of phospholipids by sPLA2.