Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation.

Shyamal P Waghwala, Taylor Ngo, Ainsely Verkaik

Methods in molecular biology (Clifton, N.J.) 2024

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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). © 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Citation

Shyamal P Waghwala, Taylor Ngo, Ainsely Verkaik. Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation. Methods in molecular biology (Clifton, N.J.). 2024;2816:129-138