Abstract：
Phospholipase D (PLD) is an enzyme with many functions, one of which is the synthesis of phosphatidic acid (PA), a molecule with a myriad of effects on various organ systems and processes. These numerous roles make it hard to understand the true action of PA in cellular and bodily processes. Imaging PLD activity is one way to better understand the synthesis of PA and start to elucidate its function. However, many of the current imaging techniques for PLD come with limitations. This chapter presents a thorough methodology of a new imaging technique for PLD activity with clickable alcohols via transphosphatidylation (IMPACT) and Real-Time IMPACT (RT-IMPACT) that takes advantage of clickable chemistry to overcome current limitations. Using strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron-demand Diels-Alder (IEDDA), and the synthesis of various organic compounds, this chapter will explain a step-by-step procedure of how to perform the IMPACT and RT-IMPACT method(s).
摘要：
磷脂酶D(PLD)是一种具有多种功能的酶。其中之一是磷脂酸（PA）的合成，对各种器官系统和过程有无数影响的分子。这些众多的角色使得难以理解PA在细胞和身体过程中的真正作用。成像PLD活性是更好地理解PA的合成并开始阐明其功能的一种方法。然而,目前PLD的许多成像技术都有局限性。本章介绍了一种通过转磷脂化（IMPACT）和实时IMPACT（RT-IMPACT）对可点击醇进行PLD活性的新成像技术的全面方法，该技术利用可点击化学来克服当前的局限性。使用菌株促进的叠氮化物-炔环加成(SPAAC)，反电子需求狄尔斯-阿尔德(IEDDA)，以及各种有机化合物的合成，本章将解释如何执行IMPACT和RT-IMPACT方法的分步过程。
