Brown H Alex, Henage Lee G, Preininger Anita M, Xiang Yun, Exton John H
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Methods Enzymol. 2007;434:49-87. doi: 10.1016/S0076-6879(07)34004-4.
Phospholipase D (PLD) is distributed widely in nature, being present in various isoforms in bacteria, protozoa, fungi, plants, and animals. It catalyzes the hydrolysis of phospholipids, primarily phosphatidylcholine (PC), into phosphatidic acid (PA) and the head group, choline. It also catalyzes a transphosphatidylation reaction in which water is replaced by a primary alcohol to yield a phosphatidyl alcohol. This reaction is exclusive to PLD and is employed as a specific assay for the enzyme in in vivo systems. When the purified enzyme is assayed in vitro, the release of choline from PC can be utilized. This chapter describes production of a recombinant mammalian isozyme of PLD (PLD1) in baculovirus-infected insect cells and its purification. It also provides details of the assay procedure in the presence and absence of regulatory proteins in vitro. The assay of the enzyme in cells in vivo is also documented using labeling of endogenous PC by incubating the cells with (3)H-labeled fatty acid. Details of the assay utilizing the transphosphatidylation reaction are presented. In this, 1-butanol is employed as the primary alcohol and [(3)H]phosphatidylbutanol is isolated by thin-layer chromatography of lipid extracts from the cells. A variation of this assay is described using deuterated 1-butanol (1-butanol-d(10)) and detection of the synthesized deuterated phosphatidylbutanol species by mass spectrometry. Convenient alternative assays for PLD and diacylglycerol (DAG) lipase activity based on fluorescence are also described. Many of the materials for these assays are available commercially, with the exception of the fluorescently labeled DAG substrate, which can be synthesized enzymatically in a simple one-step procedure.
磷脂酶D（PLD）广泛分布于自然界，在细菌、原生动物、真菌、植物和动物中以多种同工型存在。它催化磷脂（主要是磷脂酰胆碱，PC）水解为磷脂酸（PA）和头部基团胆碱。它还催化转磷脂酰基反应，其中水被伯醇取代，生成磷脂醇。该反应是PLD特有的，在体内系统中用作该酶的特异性测定方法。在体外测定纯化的酶时，可以利用PC中胆碱的释放。本章描述了在杆状病毒感染的昆虫细胞中生产重组哺乳动物PLD同工酶（PLD1）及其纯化方法。它还提供了在体外有无调节蛋白存在下测定程序的详细信息。还记录了通过用（3）H标记的脂肪酸孵育细胞对内源性PC进行标记来在体内细胞中测定该酶的方法。介绍了利用转磷脂酰基反应进行测定的详细信息。在该方法中，使用1-丁醇作为伯醇，并通过对细胞脂质提取物进行薄层层析来分离[（3）H]磷脂酰丁醇。描述了该测定方法的一种变体，使用氘代1-丁醇（1-丁醇-d（10））并通过质谱检测合成的氘代磷脂酰丁醇种类。还描述了基于荧光的PLD和二酰基甘油（DAG）脂肪酶活性的便捷替代测定方法。这些测定方法所需的许多材料都可以从商业渠道获得，但荧光标记的DAG底物除外，它可以通过简单的一步酶促合成法制备。
