A fluorescence and HPLC-based activity assay system for lipid-modifying enzymes: Lipin/Pah phosphatidic acid...

Quantitating the enzymatic activity of lipid-modifying enzymes is a critical aspect of their study. Here, we describe a general approach to measure the activity of lipid-modifying enzymes using fluorescently labeled lipid substrates. This approach uses high-performance liquid chromatography (HPLC) to separate the resulting fluorescent lipid product(s) of enzymatic catalysis from the fluorescent lipid substrates, and to concurrently quantify the products using a fluorescence detector. Here, we focus on a version of this assay that has been optimized for lipin/Pah phosphatidic acid phosphatases, a class of enzymes that catalyze the magnesium-dependent hydrolysis of phosphatidic acid into diacylglycerol. Details for delivery of a fluorescent lipid substrate to an enzyme in detergent mixed-micelles and liposomes are provided. We also describe methods to purify recombinant lipin/Pah phosphatidic acid phosphatases using E. coli as an overexpression system, which can present challenges given the role of phosphatidic acid as a central precursor in bacterial phospholipid synthesis. Overall, the fluorescence and HPLC-based activity assay described here is generally applicable to any lipid modifying enzyme and additionally allows for the detection of unsuspected products that would remain undetected by conventional methods.