Identity between the Ca2+-independent phospholipase A(2) enzymes from P388D(1) macrophages and Chinese hamster ovary cells
Identity between the Ca2+-independent phospholipase A(2) enzymes from P388D(1) macrophages and Chinese hamster ovary cells
复制标题
DOI：
10.1074/jbc.272.13.8576
复制
发表时间：
1997-03-28
影响因子：
4.8
通讯作者：
Dennis, EA
中科院分区：
文献类型：
--
作者：
Balboa, MA;Balsinde, J;Dennis, EA
最近从P388 D（1）巨噬细胞中纯化并表征了一种新的不依赖Ca 2+的磷脂酶A（2）（iPLA（2））（Ackermann，E. J，，Kempner，E，S，，and Dennis，E，A.（1994）J，Biol.Chem.269，9227-9233），这种酶似乎在调节基础磷脂重塑反应中起关键作用。来自中国仓鼠卵巢（CHO）细胞的iPLA（2）也已被纯化、分子克隆和表达（Tang，J，Kriz，R，Wolfman，N，Shaffer，M，Seehra，J，和Jones，S。S，（1997）J.Biol.Chem.272，8567-8575），我们在此报道了克隆的CHO iPLA（2）等同于从P388 D（1）细胞纯化的小鼠酶，使用基于CHO iPLA（2）序列的引物对来自P388 D细胞的cDNA片段进行聚合酶链式反应扩增，揭示了小鼠和仓鼠酶在核苷酸和氨基酸水平上的高度同源性（分别为92%和95%）。通过免疫化学、药理学和生物化学方法进一步证明了这两种蛋白质之间的同一性。因此，针对CHO酶产生的抗血清识别P388 D（1）细胞酶，并给出相似的分子量此外，CHO酶对先前显示抑制P388 D（1）酶的多种化合物的抑制具有完全相同的敏感性，所述化合物包括溴烯醇内酯，棕榈酰三氟甲基酮，此外，CHO酶被[H-3]溴烯醇内酯共价修饰依赖于活性酶，P588 D（1）iPLA（2）也是如此。最后，两种酶在相同的实验条件下具有相同的比活性。
A novel Ca2+-independent phospholipase A(2) (iPLA(2)) has recently been purified and characterized from P388D(1) macrophages (Ackermann, E. J,, Kempner, E, S,, and Dennis, E, A. (1994) J, Biol. Chem. 269, 9227-9233), This enzyme appears to play a key role in regulating basal phospholipid remodeling reactions, Also an iPLA(2) from Chinese hamster ovary (CHO) cells has been purified, molecularly cloned, and expressed (Tang, J,, Kriz, R,, Wolfman, N,, Shaffer, M,, Seehra, J,, and Jones, S. S, (1997) J. Biol. Chem. 272, 8567-8575), We report herein that the cloned CHO iPLA(2) is equivalent to the mouse enzyme purified from P388D(1) cells, Polymerase chain reaction amplification of cDNA fragments from P388D, cells using primers based on the CHO iPLA(2) sequence, revealed a high degree of homology between the mouse and hamster enzymes at both the nucleotide and amino acid levels (92 and 95%, respectively). Identity between the two proteins was further demonstrated by using immunochemical, pharmacological, and biochemical approaches. Thus, an antiserum generated against the CHO enzyme recognized the P388D(1) cell enzyme and gave similar molecular masses (about 83 kDa) for the two enzymes under the same experimental conditions, Further, the CHO enzyme has exactly the same sensitivity to inhibition by a variety of compounds previously shown to inhibit the P388D(1) enzyme, including bromoenol lactone, palmitoyl trifluoromethyl ketone, and methyl arachidonyl fluorophosphonate, Additionally, covalent modification of the CHO enzyme by [H-3]bromoenol lactone is dependent on active enzyme as is the P588D(1) iPLA(2), Finally, both enzymes have the same specific activities under identical experimental conditions.