Purification and properties of a phosphatidic acid-preferring phospholipase A(1) from bovine testis - Examination of the molecular basis of its activation
Purification and properties of a phosphatidic acid-preferring phospholipase A(1) from bovine testis - Examination of the molecular basis of its activation
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DOI：
10.1074/jbc.271.18.10874
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发表时间：
1996-05-03
影响因子：
4.8
通讯作者：
Glomset, JA
中科院分区：
文献类型：
--
作者：
Higgs, HN;Glomset, JA
我们最近在牛脑和睾丸中发现了一种胞质磷脂酶A（1）活性，它优先水解磷脂酸底物。我们还表明，该酶在Triton X-100混合胶束测定系统中显示出对磷脂酸底物的S形动力学（Higgs，H. N.，和Glomset，J. A.等人（1994）Proc. Acad. Sci.联合S. A. 91，9574-9578）。在本工作中，我们将牛睾丸酶纯化14，000倍，并使用尺寸排阻色谱法、用磷脂酶A抑制剂花生四烯酰氟膦酸甲酯标记和SDS-聚丙烯酰胺凝胶电泳的组合，以提供它是110-kDa亚基的同源四聚体的证据。对Triton胶束中酶反应的分子基础的研究表明：（a）磷脂酸的一种不可水解的sn-1-烷基-2-油酰基类似物以S形方式激活酶30倍（希尔系数3.2，EC（50）4摩尔%），而基本上不影响其对特异性二酰基磷酸甘油酯底物的偏好，（B）活化剂促进酶与胶束的紧密结合，和（c）酶对不饱和磷脂酸底物的活性受脂肪酰基链双键的位置和性质的影响。
We recently identified a cytosolic phospholipase A(1) activity in bovine brain and testis that preferentially hydrolyzes phosphatidic acid substrates. We also showed that the enzyme displays sigmoidal kinetics toward phosphatidic acid substrates in a Triton X-100 mixed micelle assay system (Higgs, H. N., and Glomset, J. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9574-9578), In the present work we purified the bovine testis enzyme 14,000 fold and used a combination of size exclusion chromatography, labeling with the phospholipase A inhibitor, methyl arachidonyl fluorophosphonate, and SDS-polyacrylamide gel electrophoresis to provide evidence that it is a homotetramer of 110-kDa subunits. Studies of the molecular basis of the enzyme reaction in Triton micelles revealed that (a) a nonhydrolyzable sn-1-alkyl-2-oleoyl analogue of phosphatidic acid activated the enzyme 30-fold in a sigmoidal fashion (Hill coefficient 3.2, EC(50) 4 mol %) without substantially affecting its preference for specific diacyl phosphoglyceride substrates, (b) the activator promoted tight binding of the enzyme to micelles, and (c) the enzyme's activity toward unsaturated phosphatidic acid substrates was affected by the location and nature of the fatty acyl chain double bonds.