Jain M K, Yu B Z, Kozubek A
Department of Chemistry, University of Delaware, Newark 19716.
Biochim Biophys Acta. 1989 Mar 27;980(1):23-32. doi: 10.1016/0005-2736(89)90195-8.
Catalytic action of phospholipase A2 is appreciably influenced by the organization and dynamics of bilayers of glycerophosphocholines (Apitz-Castro et al. (1988) Biochim. Biophys. Acta 688, 341-348). However, such effects of the quality of the interface are not observed with bilayers of glycerophosphoryl methanol and other anionic phospholipids (Jain et al. (1986) Biochim. Biophys. Acta 860, 435-447). Such differences between the catalytic susceptibility of zwitterionic versus anionic bilayers are due to a large difference in the affinity of the enzyme for these interfaces. Binding to phospholipase A2 to zwitterionic interfaces can be promoted in the presence of certain anionic additives. For example in the pre-steady-state phase of hydrolysis, segregation of the nacently produced products of hydrolysis could promote binding of phospholipase A2 to regions of higher anionic charge density in the zwitterionic interface. In this paper we show that the dynamics of segregation of the nacently produced products of hydrolysis in zwitterionic bilayers can be readily followed by monitoring the fluorescence intensity of the cationic dye NK-529 (Yu and Jain (1989) Biochim. Biophys. Acta 980, 15-22). The fluorescence emission characteristics of NK-529 change appreciably due to self-quenching of the bound dye molecules as the fatty acid molecules segregate in the bilayer. The kinetics of segregation of fatty acids during the course of hydrolysis of bilayers of zwitterionic phospholipids by phospholipase A2 exhibits an unequivocal correlation with a variety of phenomena that are observed during the transition from the pre-steady-state phase to the steady-state phase of hydrolysis in the reaction progress curves as a function of temperature and in the presence of lipophilic additives.
磷脂酶A2的催化作用受到甘油磷酸胆碱双层膜的组织和动力学的显著影响（阿皮茨 - 卡斯特罗等人（1988年），《生物化学与生物物理学报》688卷，341 - 348页）。然而，在甘油磷酸甲醇双层膜和其他阴离子磷脂双层膜中未观察到这种界面性质的影响（贾恩等人（1986年），《生物化学与生物物理学报》860卷，435 - 447页）。两性离子双层膜与阴离子双层膜催化敏感性的这种差异是由于酶对这些界面的亲和力存在很大差异。在某些阴离子添加剂存在的情况下，可以促进磷脂酶A2与两性离子界面的结合。例如，在水解的预稳态阶段，新生水解产物的分离可以促进磷脂酶A2与两性离子界面中阴离子电荷密度较高区域的结合。在本文中，我们表明，通过监测阳离子染料NK - 529的荧光强度，可以很容易地追踪两性离子双层膜中新生水解产物的分离动力学（于和贾恩（1989年），《生物化学与生物物理学报》980卷，15 - 22页）。随着脂肪酸分子在双层膜中分离时，结合的染料分子发生自猝灭，NK - 529的荧光发射特性发生明显变化。在磷脂酶A2催化两性离子磷脂双层膜水解过程中，脂肪酸分离的动力学与在反应进程曲线中从水解的预稳态阶段转变为稳态阶段期间观察到的各种现象表现出明确的相关性，这些现象是温度和存在亲脂性添加剂的函数。
