Characterization of Ca2+-dependent phospholipase A2 activity during zebrafish embryogenesis
Characterization of Ca2+-dependent phospholipase A2 activity during zebrafish embryogenesis
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DOI：
10.1074/jbc.274.27.19338
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发表时间：
1999-07-02
影响因子：
4.8
通讯作者：
Halpern, ME
中科院分区：
文献类型：
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作者：
Farber, SA;Olson, ES;Halpern, ME
我们已经开发了一种简单的荧光检测磷脂酶A（2）（PLA（2））的活性在斑马鱼胚胎，利用荧光磷脂酰胆碱底物。通过结合选择性PLA（2）抑制剂和蛋白质印迹分析，我们确定了斑马鱼胚胎发生中的主要活性为Ca（2+）依赖性胞质PLA（2）（cPLA（2））亚型的特征。胚胎cPLA（2）活性从1-细胞期一直保持恒定，直到体节发生开始，此时活性急剧增加。这种增加之前是先前鉴定的斑马鱼cPLA（2）同源物的表达（Nalefski，E.，苏茨曼湖Martin，D.，克里兹河，Towler，P.，Knopf，J.，和Clark，J.（1994）J.Biol.Chem.269，18239-18249）。通过使用淬灭的BODIPY标记的磷脂酰胆碱（仅在被PLA裂解时发出荧光）（2），在活胚胎细胞中观察到脂肪酶活性，脂肪酶活性位于核周膜。
We have developed a simple fluorescent assay for detection of phospholipase A(2) (PLA(2)) activity in zebrafish embryos that utilizes a fluorescent phosphatidylcholine substrate. By using this assay in conjunction with selective PLA(2) inhibitors and Western blot analysis, we identified the principal activity in zebrafish embryogenesis as characteristic of the Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)) subtype. Embryonic cPLA(2) activity remained constant from the 1-cell stage until the onset of somitogenesis, at which time it increased sharply. This increase was preceded by the expression of a previously identified zebrafish cPLA(2) homologue (Nalefski, E., Sultzman, L., Martin, D., Kriz, R., Towler, P., Knopf, J., and Clark, J. (1994) J. Biol. Chem. 269, 18239-18249). By using a quenched BODIPY-labeled phosphatidylcholine that fluoresces only upon cleavage by PLA(2), lipase activity was visualized in the cells of living embryos where it localized to perinuclear membranes.