Goedhart Joachim, Gadella Theodorus W J
Laboratory for Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.
Biochemistry. 2004 Apr 13;43(14):4263-71. doi: 10.1021/bi0351460.
Phosphatidic (PtdOH) acid formation is recognized as an important step in numerous signaling pathways in both plants and mammals. To study the role of this lipid in signaling pathways, it is of major interest to be able to increase the amount of this lipid directly. Therefore, "caged" PtdOH was synthesized, which releases the biologically active PtdOH upon exposure to UV. Analysis of the product revealed that two 2-nitrophenylethyl (NPE) caging groups were coupled to the phosphate headgroup of PtdOH. To measure the quantum efficiency of uncaging, a fluorimetric assay, based on the notion that the NPE cage is an efficient quencher of pyrene fluorescence, was developed. Consequently, after NPE-caged PtdOH and (N-pyrene)-PtdEtn had been mixed in DOPC vesicles, the extent of photolysis of caged PtdOH can be quantified by monitoring the increase in pyrene fluorescence. Using this assay, a quantum yield of 9.6% was determined for the uncaging reaction. The swimming green alga Chlamydomonas moewusii deflagellates upon addition of PtdOH. This response was used to study the release of PtdOH in vivo. Algae incubated with caged PtdOH only arrested swimming after exposure to UV, indicative of PtdOH release. This effect was not observed in the absence of the caged compound or when a control caged compound (caged acetic acid) was added. Fluorescein diacetate staining was used to show that the cells remained viable after UV exposure. The anticipated effect of PtdOH release is confirmed by phase contrast images of UV-exposed algae showing excision of flagella. Together, these results show that caged PtdOH can be used to efficiently increase PtdOH levels, demonstrating that it is a promising precursor for studying PtdOH-dependent signaling.
磷脂酸（PtdOH）的形成被认为是植物和哺乳动物众多信号通路中的重要一步。为了研究这种脂质在信号通路中的作用，能够直接增加这种脂质的量成为了主要关注点。因此，合成了“笼形”PtdOH，其在暴露于紫外线时会释放出具有生物活性的PtdOH。对产物的分析表明，两个2-硝基苯乙基（NPE）笼形基团与PtdOH的磷酸头部相连。为了测量脱笼的量子效率，基于NPE笼是芘荧光的有效猝灭剂这一概念，开发了一种荧光测定法。因此，在将NPE笼形PtdOH和（N-芘）-磷脂酰乙醇胺（PtdEtn）混合到二油酰磷脂酰胆碱（DOPC）囊泡中后，可以通过监测芘荧光的增加来量化笼形PtdOH的光解程度。使用该测定法，确定脱笼反应的量子产率为9.6%。游动的绿藻莱茵衣藻在添加PtdOH后会失去鞭毛。这种反应被用于研究体内PtdOH的释放。仅用笼形PtdOH孵育的藻类在暴露于紫外线后才停止游动，这表明有PtdOH释放。在没有笼形化合物或添加对照笼形化合物（笼形乙酸）时未观察到这种效应。使用二乙酸荧光素染色表明细胞在紫外线暴露后仍保持活力。紫外线暴露的藻类的相差图像显示鞭毛脱落，证实了PtdOH释放的预期效果。总之，这些结果表明笼形PtdOH可用于有效提高PtdOH水平，证明它是研究PtdOH依赖性信号传导的有前景的前体。
