Measuring Phospholipase D Enzymatic Activity Through Biochemical and Imaging Methods.

The phospholipase D (PLD) enzymatic superfamily regulates a wide range of cell biological and physiological pathways, including platelet activation, immune responses, cancer, and spermatogenesis. The three main enzymatic actions of the superfamily entail (i) hydrolyzing membrane phospholipids (phosphatidylcholine (PC) and cardiolipin) to generate choline and the second messenger signaling lipid phosphatidic acid (PA), (ii) using ethanol to transphosphatidylate PC to generate the long-lived metabolite phosphatidylethanol, and (iii) hydrolyzing RNA transcripts to generate piRNAs, the third form of endogenous RNAi. We discuss briefly previously published methods for in vitro and in vivo detection and imaging of PA, and focus on production, purification, and in vitro endonuclease activity analysis for human PLD6, a mitochondrial-tethered isoform with roles in fertility, cancer, and neuronal homeostasis.