Callisen T H, Talmon Y
Department of Chemistry, Technical University of Denmark, Lyngby.
Biochemistry. 1998 Aug 4;37(31):10987-93. doi: 10.1021/bi980255d.
Phospholipid hydrolysis to free fatty acid and 1-lyso-phospholipid by water-soluble phospholipase A2 (PLA2) at the surface of lipid membranes exhibits a poorly understood transition from a low-activity lag phase to a burst regime of rapid hydrolysis. Understanding this kinetic phenomenon may increase our insight into the function of PLA2 under physiological conditions as well as into general interfacial catalysis. In the present study we apply for the first time cryo-transmission electron microscopy (cryo-TEM) and high-performance liquid chromatography (HPLC) to characterize the PLA2 hydrolysis of phospholipid vesicles with respect to changes in lipid composition and morphology. Our direct experimental results show that the initial reaction conditions are strongly perturbed during the course of hydrolysis. Most strikingly, cryo-TEM reveals that starting in the lag phase, vesicles become perforated and degrade into open vesicles, bilayer fragments, and micelles. This structural instability extends throughout the system in the activity burst regime. In agreement with earlier reported correlations between initial phospholipase activity and substrate morphology, our results suggest that the lag-burst phenomenon reflects a cascade process. The PLA2-induced changes in lipid composition transform the morphology which in turn results in an acceleration of the rate of hydrolysis because of a strong coupling between the PLA2 activity and the morphology of the lipid suspension.
水溶性磷脂酶A2（PLA2）在脂质膜表面将磷脂水解为游离脂肪酸和溶血磷脂，从低活性的滞后阶段到快速水解的爆发阶段的转变，目前还了解甚少。理解这一动力学现象，可能会增进我们对PLA2在生理条件下功能的认识，以及对一般界面催化的认识。在本研究中，我们首次应用冷冻透射电子显微镜（cryo-TEM）和高效液相色谱（HPLC），来表征磷脂囊泡的PLA2水解在脂质组成和形态变化方面的情况。我们的直接实验结果表明，在水解过程中初始反应条件受到强烈干扰。最引人注目的是，冷冻透射电子显微镜显示，从滞后阶段开始，囊泡就会穿孔并降解为开放囊泡、双层碎片和微团。这种结构不稳定性在活性爆发阶段贯穿整个系统。与早期报道的初始磷脂酶活性与底物形态之间的相关性一致，我们的结果表明滞后-爆发现象反映了一个级联过程。PLA2诱导的脂质组成变化改变了形态，而形态的改变又由于PLA2活性与脂质悬浮液形态之间的强耦合而导致水解速率加快。
