Bascom Palmer Eye Institute, Miami Integrative Metabolomics Research Center, University of Miami Miller School of Medicine, Miami, FL, USA.
The University of Texas at Southwestern Medical School, Dallas, TX, USA.
Methods Mol Biol. 2024;2816:129-138. doi: 10.1007/978-1-0716-3902-3_12.
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).
磷脂酶 D（PLD）是一种具有多种功能的酶，其中之一是合成磷脂酸（PA），PA 作为一种分子，对各种器官系统和过程具有多种影响。这些众多的作用使得很难理解 PA 在细胞和身体过程中的真正作用。对 PLD 活性进行成像，是一种更好地理解 PA 合成并开始阐明其功能的方法。然而，目前用于 PLD 的许多成像技术都存在局限性。本章介绍了一种通过转磷酸化（IMPACT）和实时 IMPACT（RT-IMPACT）利用可点击醇对 PLD 活性进行成像的新技术的全面方法，该方法利用点击化学克服了当前的局限性。本章将使用应变促进的叠氮-炔环加成（SPAAC）、逆电子需求 Diels-Alder（IEDDA）和各种有机化合物的合成，逐步解释如何进行 IMPACT 和 RT-IMPACT 方法。
