Gheriani-Gruszka N, Almog S, Biltonen R L, Lichtenberg D
Department of Physiology and Pharmacology, Tel Aviv University School of Medicine, Israel.
J Biol Chem. 1988 Aug 25;263(24):11808-13.
Pancreatic phospholipase A2 (PLA2)-catalyzed hydrolysis of egg yolk phosphatidylcholine (PC) in mixed PC-cholate systems depends upon composition, structure, and size of the mixed aggregates. The hydrolysis of PC-cholate-mixed micelles made of an equal number of PC and cholate molecules is consistent with a Km of about 1 mM and a turnover number of about 120 s-1. Increasing the cholate/PC ratio in the micelles results in a decreased initial velocity. Hydrolysis of cholate-containing unilamellar vesicles is very sensitive to the ratio of cholate to PC in the vesicles. The hydrolysis of vesicles with an effective cholate/PC ratio greater than 0.27 is similar to that of the mixed micelles. The time course of hydrolysis of vesicles with lower effective ratios is similar to that exhibited by pure dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles in the thermotropic phase transition region. In the latter two cases, the rate of hydrolysis increases with time until substrate depletion becomes significant. The reaction can be divided phenomenologically into two phases: a latency phase where the amount of product formed is a square function of time (P(t) = At2) and a phase distinguished by a sudden increase in activity. The parameter A, which describes the activation rate of the enzyme during the initial phase in a quantitative fashion, increases with increasing [PLA2], decreasing [PC], decreasing vesicle size, and increasing relative cholate content of the vesicles. The effect of [PLA2] and [PC] on the hydrolysis reaction is similar to that found with pure DPPC unilamellar vesicles in their thermotropic phase transition region. The effect of cholate on the hydrolysis reaction is similar to that of temperature variation within the phase transition of temperature variation within the phase transition of DPPC. These results are consistent with our previously proposed model, which postulates that activation of PLA2 involves dimerization of the enzyme on the substrate surface and that the rate of activation is directly proportional to the magnitude of lipid structural fluctuations. It is suggested that large structural fluctuations, which exist in the pure lipid system in the phase transition range, are introduced into liquid crystalline vesicles by the presence of cholate and thus promote activation of the enzyme.
胰腺磷脂酶A2（PLA2）催化的蛋黄磷脂酰胆碱（PC）在PC - 胆酸盐混合体系中的水解作用取决于混合聚集体的组成、结构和大小。由等量PC和胆酸盐分子构成的PC - 胆酸盐混合胶束的水解作用符合约1 mM的米氏常数（Km）和约120 s⁻¹的转换数。增加胶束中胆酸盐/PC的比例会导致初始速度降低。含胆酸盐的单层囊泡的水解作用对囊泡中胆酸盐与PC的比例非常敏感。有效胆酸盐/PC比例大于0.27的囊泡的水解作用与混合胶束的水解作用相似。有效比例较低的囊泡的水解时间进程与热致相变区域中纯二棕榈酰磷脂酰胆碱（DPPC）大单层囊泡所呈现的相似。在后两种情况下，水解速率随时间增加，直至底物消耗变得显著。该反应在现象学上可分为两个阶段：一个潜伏期阶段，其中形成的产物量是时间的平方函数（P(t) = At²），以及一个以活性突然增加为特征的阶段。参数A以定量方式描述酶在初始阶段的活化速率，它随着[PLA2]的增加、[PC]的降低、囊泡大小的减小以及囊泡中胆酸盐相对含量的增加而增加。[PLA2]和[PC]对水解反应的影响与在热致相变区域中纯DPPC单层囊泡的情况相似。胆酸盐对水解反应的影响与DPPC相变过程中温度变化的影响相似。这些结果与我们先前提出的模型一致，该模型假定PLA2的活化涉及酶在底物表面的二聚化，并且活化速率与脂质结构波动的幅度成正比。有人提出，在相变范围内纯脂质体系中存在的大结构波动通过胆酸盐的存在被引入液晶囊泡中，从而促进酶的活化。
