Wacklin Hanna P, Tiberg Fredrik, Fragneto Giovanna, Thomas Robert K
Oxford University, Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, UK.
Biochim Biophys Acta. 2007 May;1768(5):1036-49. doi: 10.1016/j.bbamem.2006.10.020. Epub 2006 Nov 10.
We have monitored the composition of supported phospholipid bilayers during phospholipase A(2) hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA(2) shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5+/-3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA(2), which resides in a 21+/-1 A thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d(31)-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA(2) molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA(2).
我们使用镜面中子反射和椭偏测量技术监测了磷脂酶A(2)水解过程中支持的磷脂双层的组成。猪胰磷脂酶A(2)显示出长达数小时的长延迟期，在此期间酶与双层表面结合，但在快速水解开始之前只有5±3%的脂质发生反应。位于水-双层界面21±1埃厚层中的磷脂酶A(2)的量及其向膜内的渗透深度在延迟期增加，延迟期的长度也与酶浓度成正比。对单链氘标记的d(31)-POPC的水解首次揭示，反应产物在膜和水环境之间的分布存在显著不对称。溶血脂质离开膜，而与界面结合的磷脂酶A(2)分子数量随着脂肪酸含量的增加而增加。这些结果构成了对膜结构和组成的首次直接测量，包括水解过程中酶的位置和数量。我们根据脂肪酸介导的磷脂酶A(2)激活模型对这些结果进行了讨论。
