Studies on the separation of phospholipase A_2 preferred the arachidonoyl species of phospholipids

测定了钙调蛋白拮抗剂对经调理酵母多糖处理的肺泡巨噬细胞产生游离脂肪酸的影响。在W-7和三氟拉嗪存在下，优化酵母多糖诱导的花生四烯酸(20：4)的产生被显著抑制，而对其他脂肪酸的产生没有影响。钙调素拮抗剂似乎抑制磷脂酶A2，因为W-7取消了胆碱甘油磷脂(CGP)和肌醇甘油磷脂(IGP)的20：4解离。在没有胞外Ca~(2+)的情况下，经调理的酵母多糖激发后，其20：4的解离作用显著降低，说明花生四烯酸的释放需要胞外Ca~(2+)的内流。另一方面，胞外Ca~(2+)耗竭后，调理酵母多糖对肺泡巨噬细胞释放油酸和亚油酸的作用不受抑制。磷脂酶A_2活性主要存在于肺泡巨噬细胞的胞浆中，占总活性的70%。钙能促进胞浆磷脂酶A_2对16：0~20：4GPC的水解性，但对16：0~18：1和16：0~18：2胞浆磷脂酶A_2的水解性无明显影响。目前的研究表明，兔肺泡巨噬细胞中的磷脂酶A_2受钙离子和钙调蛋白的控制，优先从磷脂中水解花生四烯酸。

The effects of calmodulin antagonists on the amount of free fatty acid produced by alveolar macrophages exposed to opsonized zymosan were determined. Opsonized zymosan-induced free arachidonic acid (20:4) production was dramatically suppressed in the presence of W-7 and trifluoperazine without an effect on the production of other fatty acids. Calmodulin antagonists appeared to inhibit phospholipase A_2, since W-7 abolished the liberation of 20:4 from choline glycerophospholipids (CGP) and inositol glycerophospholipids (IGP$. After the challenge with opsonized zymosan, the 20:4 liberation caused by opsonized zymosan was significantly reduced in the absence of extracellular Ca^<2+>, indicating that the release of arachidonic acid required the influx of the extracelluler Ca^<2+> into cell. On the other hand, oleic acid and linoleic acid liberation from alveolar macrophages exposed to opsonized zymosan was not inhibited after the depletion of extracellular Ca^<2+>.Most of phospholipase A_2 activity was present in the cytosol fraction of alveolar macrophages, representing 70% of the total activity. Calcium enhanced the hydrolysis of 16:0-20:4 GPC with cytosolic phospholipase A_2, although the phdrolysis of 16:0-18:1 and 16:0-18:2 species of CGP was not changed by calcium.The purification of cytosolic phospholipase A_2 has been performed by using the hydrophordic column, ion exchange column and gel filtration column. However, phospholipase A_2 which selectively hydrolyzed the arechidonoyl species of phospholipids failed to be isolated.The present studies indicate that phospholipase A_2 in rabbit alveolar macrophages is controlled by Ca^<2+> and calmodulin, and preferentially hydrolyzes arachidonic acid from phospholipids.