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磷脂和洗涤剂的不同组合或磷脂分散物具有最低的磷脂酶A活性。然而，在无菌条件下，在30ﾟC下孵育6小时后，依赖于钙离子的磷脂酰乙醇胺(PE)分散体的聚乳酸活性逐渐增加，并在48小时后达到最大值。这种活性的激活完全需要Ca~(2+)离子的存在。其他二价阳离子，锶和镁离子的作用顺序也是如此，但幅度小于钙离子。鸟嘌呤核苷酸、GTP、GMP、cGMP和鸟苷可提高激活速率，而ATP和cAMP则抑制激活。有趣的是，NO形成试剂NaN_3、NaNO_2和硝酸异山梨酯抑制了激活。丝氨酸蛋白酶抑制剂显著抑制了激活，提示激活机制是通过有限的磷脂酶原A2的蛋白分解。通过Q-琼脂糖层析、苯基琼脂糖层析、琼脂糖凝胶4B层析和Cosmosil 5C_8-300层析，我们将先前在4ﾟC激活的牛主动脉PLA纯化到接近均一的水平。纯化后的酶的性质如下：1.与其他类型的磷脂相比，该酶更喜欢PE分散体。阴离子洗涤剂对酶活性有抑制作用。：B，该酶的相对分子质量约为30 kDa。C、钙离子是活性所必需的。通过酶的作用，混合酰基磷脂的sn-1和sn-2位都释放出D，脂肪酸。E、牛主动脉磷脂酶A_2的免疫组织化学与I、II磷脂酶A_2不同。

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.