Effect of the structure of phospholipid on the kinetics of intravesicle scooting of phospholipase A2.
Effect of the structure of phospholipid on the kinetics of intravesicle scooting of phospholipase A2.
复制标题
磷脂结构对磷脂酶 A2 囊泡内移动动力学的影响。
DOI：
10.1016/0005-2736(86)90543-2
复制
发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Eibl,H
中科院分区：
文献类型：
--
作者：
Jain,MK;Rogers,J;Marecek,JF;Ramirez,F;Eibl,H
在不添加任何添加剂的情况下，研究了猪胰腺磷脂酶A2对50多个合成的1，2-二甘油-3-磷酸衍生物及其类似物的囊泡的作用。总体而言，磷酸盐上较短的酰基链和较小的取代基是较好的底物，而具有较大非极性取代基的磷脂不会被水解。不同磷脂的界面翻转速率常数Ki均小于0.1~1/min。在极性取代基较大的磷脂囊泡中，结合酶的囊泡间交换较快，在氯离子、硫酸盐和硫氰酸盐等阴离子的存在下，结合酶的囊泡间交换被促进。这些因素降低了酶在双层上的停留时间，从而有效地降低了水解率。在盐存在下，该酶在阴离子磷脂界面上的表观Km在40~100μM范围内，比Trp-3荧光增强法测得的结合酶的解离常数大3~7倍。结合酶在不同脂类的囊泡中的量子产率被发现有高达4倍的差异。推测这种差异是由于E∗+S到E∗S的平衡，其中E∗S具有较高的荧光强度。讨论了钙在阴离子界面生成酶结合位中的作用，阴离子锚定位在酶上的作用，以及催化效率与荧光量子产率的关系。
Action of pig pancreatic phospholipase A2on vesicles of over 50 synthetic 1,2-diacylglycero-3-phosphate derivatives and analogs is examined in the absence of any additives. In general, shorter acyl chains and small substituents on the phosphate make a better substrate, while phospholipids with large apolar substituents are not hydrolyzed. The interfacial turnover rate constant for scooting kinetics,ki, for the various phospholipids were from less than 0.1 to 1 per min. Intervesicle exchange of the bound enzyme is faster in vesicles of phospholipids with larger polar substituents, and it is promoted in the presence of anions like chloride, sulfate and thiocyanate. These factors lower the residence time of the enzyme on the bilayer and therefore effectively decrease the rate of hydrolysis. The apparentKmfor the enzyme in the interface of anionic phospholipids in the presence of salts is in the 40 to 100 μM range which is 3- to 7-times larger than the dissociation constants for the bound enzyme measured by fluorescence enhancement of Trp-3. The quantum yield of the bound enzyme in vesicles of the various lipids is found to be up to 4-fold different. It is suggested that this difference is due to the E∗+ S to E∗S equilibrium, where E∗S has higher fluorescence intensity. The role of calcium in generating the enzyme binding site at the anionic interface, the role of anion anchoring site on the enzyme, and the relationship between the catalytic efficiency and the fluorescence quantum yields are discussed.