测定磷脂酶A2活性。
Assaying phospholipase A2 activity.
作者信息
Leslie Christina C, Gelb Michael H
机构信息
Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO, USA.
出版信息
Methods Mol Biol. 2004;284:229-42. doi: 10.1385/1-59259-816-1:229.
Mammalian cells contain many structurally and functionally diverse phospholipases A2 (PLA2) that catalyze the hydrolysis of sn-2 fatty acid from membrane phospholipid. Assays are described for measuring the activity of Group IVA cytosolic PLA2alpha(cPLAalpha) and for secreted PLA2s (sPLA2) that are suitable for purified enzymes and for measuring activity in crude cell lysates and culture medium. The assay for cPLA2alpha involves measuring the calcium-dependent release of radiolabeled sn-2 arachidonic acid from small unilamellar vesicles of phosphatidylcholine. Methods are described for distinguishing cPLA2alpha activity in cell lysates from other PLA2s. sPLA2 activity is monitored using a fluorimetric assay that measures the continuous calcium-dependent formation of albumin-bound pyrene fatty acid from the sn-2 position of phosphatidylglycerol.
哺乳动物细胞含有许多结构和功能各异的磷脂酶A2（PLA2），它们催化膜磷脂中sn-2脂肪酸的水解。本文描述了用于测量IVA组胞质磷脂酶A2α（cPLAα）和分泌型磷脂酶A2（sPLA2）活性的测定方法，这些方法适用于纯化的酶，也适用于测量粗细胞裂解物和培养基中的活性。cPLA2α的测定涉及测量从磷脂酰胆碱小单层囊泡中钙依赖性释放放射性标记的sn-2花生四烯酸。文中还描述了区分细胞裂解物中cPLA2α活性与其他PLA2活性的方法。sPLA2活性通过荧光测定法进行监测，该方法测量从磷脂酰甘油的sn-2位置连续钙依赖性形成与白蛋白结合的芘脂肪酸。
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