BINDING OF PHOSPHOLIPASE A2 TO BILAYERS

磷脂酶A_2对磷脂双分子层的作用及其生物活性 膜依赖于它们的组织。 本实验的目的 研究的目的是描述这种相互作用的控制因素， 含有二酰基磷脂酰胆碱、溶血磷脂酰胆碱和 脂肪酸 作为第一步，酶与双层的结合将是 通过荧光增强详细研究。 绑定数据是 最简单的平衡反应E加上Ln小于 大于ELn，ELn表示第一个预稳态步骤， 酶的催化循环，并且该步骤之后是ELn加S减 S大于ELn加上P。从结合等温线，我们 获得解离常数和N，脂质分子的数量， 与一个酶分子结合 表观解离常数 （等于N.Kd）预期与表观动力学有关 米氏常数，Km。 因此，我们建议测量约束力， 在可比条件下的动力学参数，以评估 脂质成分和温度。 我们将比较结合和动力学 作为脂质组分（链）结构的函数的参数 长度、不饱和度、头基变化）。 此信息将 与混合脂质分散体的相性质相关， 为了评估由于横向相位引起的相位边界的作用， 分离为可能的磷脂酶A2结合位点。 等 实验预计将提供所需的基本信息， 磷脂酶A2的界面活化机制。 激活 磷脂酶A2被认为是第一个和限速步骤， 在体内的生物合成，因为作用 磷脂酶A2从适当的磷脂中释放花生四烯酸。 所有这些研究都将在来自猪胰腺的磷脂酶A2上进行， 并且以后可以扩展到来自其他来源的酶。

Action of phospholipase A2 on phospholipid bilayers and biological membranes depends upon their organization. The experimental aim of this study is to characterize the factors governing such interactions in bilayers containing diacylphosphatidylcholine, lysophosphatidylcholine, and fatty acid. As a first step, binding of the enzyme to the bilayers will be studied in detail by fluorescence enhancement. The binding data is to be quantitated for the simplest equilibrium reaction E plus Ln less than greater than ELn, which represents the first presteady-state step in the catalytic cycle of the enzyme, and this step is followed by ELn plus S less than greater than Eln.S greater than ELn plus P. From binding isotherms we obtain the dissociation constant and N, the number of lipid molecules that bind to one enzyme molecule. The apparent dissociation constant (equalN.Kd) is expected to be related to the apparent kinetic Michaelis-Menten constant, Km. Thus we propose to measure the binding and kinetic parameters under comparable conditions to evaluate the effect of lipid composition and temperature. We will compare the binding and Kinetic parameters as a function of the structure of the lipid components (chain length, unsaturation, head group variation). This information will be correlated with the phase properties of the mixed lipid dispersions in order to evaluate the role of phase boundaries due to lateral phase separation as the possible phospholipase A2 binding sites. Such experiments are expected to provide basic information needed to elaborate the mechanism of interfacial activation of phospholipase A2. Activation of phospholipase A2 is thought to be the first and the rate limiting step in the biosynthesis of prostaglandins in vivo, since the action of phospholipase A2 releases arachidonic acid from appropriate phospholipids. All these studies will be conducted on phospholipase A2 from pig pancreas, and could be later extended to the enzyme from other sources.