Enzymatic properties of a phospholipase A_2 from vascular smooth muscles and its physiological significance

1. 新鲜制备的牛主动脉提取物对不同的磷脂和洗涤剂或磷脂分散体的各种组合具有最低的磷脂酶a （PLA）活性。然而，在30°C的无菌条件下，提取物对磷脂酰乙醇胺（PE）分散体的Ca^<2+>依赖性PLA活性在延迟6小时后逐渐增加，并在48小时后达到最大值。PLA活性的激活绝对需要Ca^<2+>离子的存在。其他二价阳离子，Sr^<2+>和Mg^<2+>也在这个顺序上有效，但作用程度不如Ca^<2+>。鸟嘌呤核苷酸、GTP、GMP、cGMP和鸟苷提高了激活率，而ATP和cAMP抑制了激活率。有趣的是，no形成试剂NaN_3， NaNO_2和硝酸异山梨酯抑制了活化。丝氨酸蛋白酶抑制剂明显抑制了激活，提示激活机制是通过抑制失活的前磷脂酶A.2的蛋白水解。通过Q-Sepharose、phenyl-Sepharose、Sepharose 4B和Cosmosil 5C_8-300层析，我们纯化了先前在4 C活化的牛主动脉PLA，得到了接近均匀性的PLA。纯化后的酶具有以下特征：a，该酶比其他种类的磷脂更喜欢PE分散体。阴离子洗涤剂抑制酶活性。b，酶的分子量约为30kda。c、钙离子是维持活性所必需的。d，在酶的作用下，脂肪酸从混合酰基磷脂的sn-1和sn-2位点释放。e、牛主动脉PLA在免疫化学上与ⅰ组和ⅱ组有明显差异。

1. Freshly prepared extracts of bovine aorta contained a minimal phospholipase A (PLA) activity towards either various combinations of different phospholipids and detergents or phospholipid dispersions. However, incubation of the extracts at 30ﾟC under sterile conditions led to a gradual increase in Ca^<2+>-dependent PLA activity towards phosphatidylethanolamine (PE) dispersions after a 6-hour lag, and it reached a maximum after 48h. This activation of PLA activity absolutely required the presence of Ca^<2+> ions. Other divalent cations, Sr^<2+> and Mg^<2+> also were effective in this order, but to a less extent than Ca^<2+> ions. Guanine nucleotides, GTP, GMP, cGMP, and guanosine, enhanced the activation rate, whereas ATP and cAMP suppressed it. Interestingly, NO-forming reagents, NaN_3, NaNO_2, and isosorbide dinitrate, inhibited the activation. Serine protease inhibitors markedly inhibited the activation, suggesting the activation mechanism through the limited proteolysis of an inactive prophospholipase A.2. We purified to near homogeneity bovine aorta PLA, which had been previously activated at 4ﾟC, through the sequential use of Q-Sepharose, phenyl-Sepharose, Sepharose 4B, and Cosmosil 5C_8-300 chromatographies. The purified enzyme was characterized as follows : a, The enzyme prefer PE dispersions over other classes of phospholipid. Anionic detergent inhibited the enzyme activity. : b, The molecular mass of the enzyme was found to be about 30 kDa. c, Calcium ions were essential for activity. d, Fatty acids were released from both sn-1 and sn-2 positions of mixed acyl phospholipids by the enzyme action. e, Bovine aorta PLA was immunochemically distinct from group I, and II phospholipases A_2.