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

研究了钙调素拮抗剂对活化酶酶体的肺泡巨噬细胞产生游离脂肪酸的影响。在W-7和三氟拉嗪的存在下，Opsonized zymosa诱导的游离花生四烯酸（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A_2，因为W-7消除了从胆碱甘油磷脂（CGP）和肌醇甘油磷脂（IGP$）中释放的20:4。在没有细胞外Ca^<2+>的情况下，经活化的酶酶引起的20:4的释放率显著降低，这表明花生四烯酸的释放需要细胞外Ca^<2+>流入细胞内。另一方面，在细胞外Ca^<2+>耗竭后，暴露于调理酶蛋白的肺泡巨噬细胞的油酸和亚油酸释放不受抑制。磷脂酶A_2活性主要存在于肺泡巨噬细胞的胞浆部分，占总活性的70%。钙促进了胞质磷脂酶A_2对16:0 ~ 20:4 GPC的水解，但对16:0 ~ 18:1和16:0 ~ 18:2 CGP的水解没有影响。采用疏水柱、离子交换柱和凝胶过滤柱对胞质磷脂酶A_2进行了纯化。但未能分离到选择性水解槟子油酰基磷脂的磷脂酶A_2。目前的研究表明，兔肺泡巨噬细胞的磷脂酶A_2受Ca^<2+>和钙调素的控制，并优先水解磷脂中的花生四烯酸。

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.