Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University & Oncode Institute, RA, Leiden, The Netherlands.
Methods Mol Biol. 2023;2576:233-240. doi: 10.1007/978-1-0716-2728-0_19.
N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the principal enzyme that generates N-acylethanolamines (NAEs), a family of signaling lipids that includes the endocannabinoid anandamide. To investigate the biological function and biosynthesis of NAEs, we sought to develop potent NAPE-PLD inhibitors. To this aim, we utilized a high-throughput screening-compatible NAPE-PLD activity assay, which uses the fluorescence-quenched substrate PED6. This assay conveniently uses membrane fractions of NAPE-PLD overexpressing HEK293T cell lysates, thus avoiding the need for protein purification. Here, we give a detailed description of the NAPE-PLD PED6 fluorescence activity assay, which has increased throughput compared to previous radioactivity- or mass-spectrometry-based assays.
N-酰基磷酰乙醇胺磷脂酶 D（NAPE-PLD）被认为是生成 N-酰基乙醇胺（NAEs）的主要酶，NAEs 是一类信号脂质，包括内源性大麻素大麻素。为了研究 NAEs 的生物学功能和生物合成，我们试图开发有效的 NAPE-PLD 抑制剂。为此，我们利用了一种高通量筛选兼容的 NAPE-PLD 活性测定法，该测定法使用荧光猝灭底物 PED6。该测定法方便地使用了过表达 NAPE-PLD 的 HEK293T 细胞裂解物的膜部分，从而避免了蛋白质纯化的需要。在这里，我们详细描述了 NAPE-PLD PED6 荧光活性测定法，与以前基于放射性或质谱的测定法相比，该方法的通量更高。
