Phospholipase A2 activity in resting and activated human neutrophils. Substrate specificity, pH dependence, and subcellular localization.
Phospholipase A2 activity in resting and activated human neutrophils. Substrate specificity, pH dependence, and subcellular localization.
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静息和激活的人中性粒细胞中的磷脂酶 A2 活性。
DOI：
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发表时间：
1988
影响因子：
4.8
通讯作者：
F. Mollinedo
中科院分区：
文献类型：
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作者：
Jesús Balsinde;E. Diez;A. Schüller;F. Mollinedo
我们已经研究了磷脂酶A2的活性分级人类中性粒细胞，采用标记的磷脂酰肌醇，磷脂酰胆碱，磷脂酰乙醇胺作为外源性底物。我们使用这些标记在sn-1位置的磷脂底物，并测量所得的标记溶血磷脂形式，以确定磷脂酶A2的特异性。在核后上清液从休息和A23187激活的细胞，磷脂酶A2的活性表现出类似的pH依赖性曲线与两个pH最适在5.5和7.5。活化细胞的提取物显示酶活性增加3-6倍。通过在连续蔗糖梯度上分离核后上清液，研究了静息和A23187处理的人中性粒细胞中磷脂酶A2活性的亚细胞分布。中性磷脂酶A2表现为膜结合酶，主要定位于质膜，嗜天青颗粒，并在一个不明确的区域之间的特定颗粒和线粒体的梯度。磷脂酶A2位于这个不确定的区域显示出更高程度的激活比位于A23187处理的细胞中的其他亚细胞颗粒。在细胞刺激过程中细胞内磷脂酶A2活性的这种特异性活化表明细胞区室化可能通过产生花生四烯酸代谢物在细胞活化剂和/或信号转导剂的形成中起作用。磷脂酰肌醇是一个更好的底物质膜酶，而磷脂酰胆碱和磷脂酰乙醇胺表现为更好的底物细胞内细胞器磷脂酶A2的活动。磷脂酶A2在pH 5.5时活性最大，表现为可溶性酶，几乎完全定位于嗜天青颗粒中。在细胞活化后，这种酸性酶活性以与β-葡萄糖醛酸苷酶（嗜天青颗粒的标记物）类似的方式释放。这些结果表明磷脂酶A2活性的不同分子特性，其细胞位置的基础上。
We have studied the phospholipase A2 activity in fractionated human neutrophils, employing labeled phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine as exogenous substrates. We used these phospholipid substrates labeled in the sn-1 position and measured the resulting labeled lysophospholipid forms in order to ascertain the phospholipase A2 specificity. In postnuclear supernatants from resting and A23187-activated cells, the phospholipase A2 activity showed a similar pH dependence curve with two pH optima at 5.5 and 7.5. Extracts from activated cells showed a 3-6-fold increase in enzyme activity. The subcellular distribution of phospholipase A2 activity in resting and A23187-treated human neutrophils was investigated by fractionation of postnuclear supernatants on continuous sucrose gradients. The neutral phospholipase A2 behaved as a membrane-bound enzyme and was mainly localized in the plasma membrane, the azurophilic granule, and in an ill-defined region of the gradient between the specific granules and mitochondria. The phospholipase A2 located in this undefined region showed a higher degree of activation than that located in other subcellular particulates in A23187-treated cells. This specific activation of an intracellular phospholipase A2 activity during cell stimulation indicates that cell compartmentalization may play a role in the formation of cell-activating and/or signal-transducing agents through the generation of arachidonate metabolites. Phosphatidylinositol was a better substrate for the plasma membrane enzyme, whereas phosphatidylcholine and phosphatidylethanolamine behaved as better substrates for intracellular organelle phospholipase A2 activities. The phospholipase A2 with maximal activity at pH 5.5 behaved as a soluble enzyme, and was almost completely localized in the azurophilic granules. Upon cell activation this acid enzyme activity was released in a similar way to beta-glucuronidase, a marker of azurophilic granules. These results demonstrate the different molecular properties of the phospholipase A2 activity, on the basis of its cellular location.