cPLA₂ Assay Kit

Phospholipase A₂ catalyzes the hydrolysis of fatty acids at the sn-2 position of glycerophospholipids, yielding a free fatty acid and a lysophospholipid as products.¹ The release of arachidonic acid from membrane phospholipids by these enzymes is believed to be the key step in the biosynthesis of eicosanoids.² There are primarily three different kinds of phospholipase A₂. They are secretory (sPLA₂), calcium-dependent cytosolic (cPLA₂), and calcium-independent cytosolic (iPLA₂) phospholipase A₂. Of these three different types of enzymes, only the cPLA₂ exhibits specificity towards arachidonic acid whereas all others can hydrolyze any fatty acid at the sn-2 position. Arachidonoyl Thio-PC is a substrate for cPLA₂ by virtue of the presence of arachidonic acid at the sn-2 position of the glycerophospholipid.³ Hydrolysis of the arachidonoyl thioester bond at the sn-2 position by PLA₂ releases a free thiol which can be detected by DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)). This assay can be used to determine the activity of cPLA₂ in purified preparations, cell cultures, or tissue homogenates that are known to contain only cPLA₂. Use of this assay with preparations containing more than one type of PLA₂ will result in the measurement of total PLA₂ activity rather than cPLA₂ alone. Isozyme-specific cPLA₂ activity can be measured by excluding sPLA₂ or inhibiting iPLA₂ activities in the assay. Each kit contains cPLA₂ assay buffer, DTNB/EGTA, Arachidonoyl Thio-PC (substrate), bee venom PLA₂ (control), bromoenol lactone solution, a 96 well plate, and complete instructions.

Needed but not supplied: Please download the kit booklet to verify if UltraPure Water (Milli-Q or equivalent) or any other components are needed for this assay.

WARNING This product is not for human or veterinary use.

1. Dennis, E.A. Phospholipases. The Enzymes XVI, (1983).

2. Dennis, E.A. Diversity of group types, regulation, and function of phospholipase A₂. The Journal of Biological Chemisty 269(18), 13057-13060 (1994).

3. Reynolds, L.J., Hughes, L.L., Yu, L., et al. 1-Hexadecyl-2-arachidonoylthio-2-deoxy-sn-glycero-3-phosphorylcholine as a substrate for the microtiterplate assay of human cytosolic phospholipase A₂. Anal. Biochem. 217, 25-32 (1994).

Brouwers, H., Jónasdóttir, H.S., Kuipers, M.E., et al. Anti-inflammatory and proresolving effects of the omega-6 polyunsaturated fatty acid adrenic acid. J. Immunol. 205(10), 2840-2849 (2020).

Chiu, C.-C., Yeh, T.-H., Lu, C.-S., et al. PARK14 PLA2G6 mutants are defective in preventing rotenone-induced mitobhondrial dysfunction, ROS generation and activation of mitochondrial apoptotic pathway. Oncotarget 8(45), 79046-79060 (2017).

Roy, D., Mondal, S., Khurana, A., et al. Loss of HSulf-1: The missing link between autophagy and lipid droplets in ovarian cancer. Sci. Rep. 7:41977, (2017).

Tamayose, C.I., Romoff, P., Toyama, D.O., et al. Non-clinical studies for evaluation of 8-C-rhamnosyl apigenin purified from Peperomia obtusifolia against acute edema. Int. J. Mol. Sci. 18(9), E1972 (2017).

Arana, L., Gangoiti, P., Ouro, A., et al. Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate. Exp. Cell Res. 318(4), 350-360 (2012).

Ling, H., Jia, X., Zhang, Y., et al. Pachymic acid inhibits cell growth and modulates arachidonic acid metabolism in nonsmall cell lung cancer A549 cells. Mol. Carcinog. 49(3), 271-282 (2010).

Bellido Reyes, Y.A., Shimoyama, T., Akamatsu, H., et al. MCI-186 (edaravone), a free radical scavenger, attenuates ischemia-reperfusion injury and activation of phospholipase A₂ in an isolated rat lung model after 18 h of cold preservation. Eur. J. Cardiothorac. Surg. 29(3), 304-311 (2006).