Singer Alan G, Ghomashchi Farideh, Le Calvez Catherine, Bollinger James, Bezzine Sofiane, Rouault Morgane, Sadilek Martin, Nguyen Eric, Lazdunski Michel, Lambeau Gerard, Gelb Michael H
Department of Chemistry, University of Washington, Seattle 98195, USA.
J Biol Chem. 2002 Dec 13;277(50):48535-49. doi: 10.1074/jbc.M205855200. Epub 2002 Sep 30.
Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A(2) (sPLA(2)s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA(2)s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 microm range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA(2)s, with Me-Indoxam being the most generally potent sPLA(2) inhibitor. A dramatic correlation exists between the ability of the sPLA(2)s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA(2)s are uniquely efficient in this regard.
在大肠杆菌和昆虫细胞中表达全套人源和鼠源的第I、II、V、X和XII组分泌型磷脂酶A2（sPLA2），已获得了纯的重组酶，用于详细的比较界面动力学和结合研究。这组哺乳动物sPLA2对二硫苏糖醇表现出显著不同的敏感性。这些酶对不同磷脂组成的囊泡水解的比活性变化高达4个数量级，但所有酶对具有不同极性头部基团的磷脂都表现出相似的催化位点特异性。对sn-2多不饱和脂肪酸与饱和脂肪酰链的区分小于6倍。根据磷脂底物的不同，这些酶对钙激活的表观解离常数在1 - 225微摩尔范围内。对一组12种活性位点导向的竞争性抑制剂的抑制作用分析表明，哺乳动物sPLA2之间的效力存在很大差异，其中甲磺酰吲哚美辛是最具普遍效力的sPLA2抑制剂。sPLA2在体外有效水解富含磷脂酰胆碱的囊泡的能力与外源添加到哺乳动物细胞时释放花生四烯酸的能力之间存在显著相关性；在这方面，第V组和第X组sPLA2具有独特的高效性。
