Grataroli R, Termine E, Portugal H, Pauli A M, Lafont H, Nalbone G
Institut National de la Santé et de la Recherche Médicale, Unité 130, Marseille, France.
Biochim Biophys Acta. 1991 Mar 12;1082(2):130-5. doi: 10.1016/0005-2760(91)90186-l.
In the present study, we have performed experiments to gain some insight into the subcellular localization and biochemical properties of gastric mucosal phospholipase A2. After classical subcellular fractionation of whole glandular stomach mucosa, we found that gastric phospholipase A2 was essentially enriched in the 105,000 x g pellet that contains microsomes and plasma membranes. Except for the cytosol, all the subcellular fractions exhibited similar phospholipase A2 activity (i.e., optimum of pH, calcium dependence, apparent Km and positional specificity). The high-speed pellet was further characterized by ultracentrifugation on a sucrose gradient. Data showed that the sedimentation profile of phospholipase A2 was quite similar to those of plasma membrane markers and more specifically to an apical membrane marker. These results, taken together, showed that a gastric phospholipase A2 is distributed among the various subcellular fractions (as a result of cross-contamination) together with the membrane fraction on which it is associated. It is proposed that this fraction is the apical plasma membrane which would be the main site of phospholipase A2 action for arachidonic acid release. Lysophospholipase showed the same sedimentation profile as phospholipase A2, whereas acyl CoA-lysophosphatidylcholine: acyltransferase mainly sedimented with heavy microsomes. The substrate specificity of the enzyme was assessed by endogenous hydrolysis of gastric mucosal phospholipids. We were able to show that the enzyme acts at nearly the same rate on two major gastric membrane phospholipids, namely phosphatidylcholine and phosphatidylethanolamine.
在本研究中，我们进行了实验，以深入了解胃黏膜磷脂酶A2的亚细胞定位和生化特性。对全腺胃黏膜进行经典的亚细胞分级分离后，我们发现胃磷脂酶A2主要富集于含有微粒体和质膜的105,000×g沉淀中。除了胞质溶胶外，所有亚细胞组分均表现出相似的磷脂酶A2活性（即pH最佳值、钙依赖性、表观Km和位置特异性）。通过在蔗糖梯度上进行超速离心对高速沉淀进行进一步表征。数据显示，磷脂酶A2的沉降图谱与质膜标志物的沉降图谱非常相似，更具体地说，与顶端膜标志物的沉降图谱相似。综合这些结果表明，胃磷脂酶A2与它所结合的膜组分一起分布在各个亚细胞组分中（由于交叉污染）。有人提出，这个组分是顶端质膜，它可能是磷脂酶A2释放花生四烯酸的主要作用位点。溶血磷脂酶的沉降图谱与磷脂酶A2相同，而酰基辅酶A-溶血磷脂酰胆碱：酰基转移酶主要与重微粒体一起沉降。通过胃黏膜磷脂的内源性水解来评估该酶的底物特异性。我们能够证明该酶对两种主要的胃膜磷脂，即磷脂酰胆碱和磷脂酰乙醇胺的作用速率几乎相同。
