Phospholipase D (PLD) activity hydrolyzes membrane phospholipids into phosphatidic acid and their respective polar headgroups. For example, phosphatidylcholine (PC) would yield phosphatidic acid (PA) and choline. While the production of choline is presumably not important to direct signaling, increases in PA can have profound consequences. PA may act either as a direct second messenger or as a precursor to other signaling molecules (diacylglycerol via a PA phosphatase activity or lysophosphatidic acid via a PA acyl hydrolase). In addition to the potential production of second messenger molecules in response to hormones, the hydrolysis of phospholipids by PLD could give rise to profound changes in the local lipid composition and physical properties of membranes. This chapter describes methods for facilitating the characterization of enzymatic activity. These include a simple assay that uses exogenous phosphatidylcholine as the substrate for the enzyme, the preparation of native ADP-ribosylation factor (ARF) from brain cytosol for use in stimulating PLD activity, and procedures for solubilizing and measuring PLD activity from cultured cells and mammalian tissues.
ASJC Scopus subject areas
- Molecular Biology