Distinguishing phospholipase A2 types in biological samples by employing group-specific assays in the presence of inhibitors

This manuscript reviews and updates radiolabel-based enzyme assays designed to distinguish the activity of phospholipase A(2) (PLA(2)) types in biological samples. This approach should be useful in lipidomics studies. The assays were originally designed to differentiate between Group IVA cytosolic PLA(2) (GIVA cPLA(2)), Group VIA calcium-independent PLA(2) (GVIA iPLA(2)), Group IIA secreted PLA(2) (GIIA sPLA(2)) and Group V secreted PLA(2) (GV sPLA(2)). The specificity of these assays has now been confirmed using purified, recombinant human PLA(2)s and the utility of these assays is demonstrated with rat spinal cord homogenate as an example of a biological tissue sample of interest to the neuroscience community. Modifications to the original assays by the addition of group-specific inhibitors are presented to ensure the specificity of the assays and to further differentiate between recently identified PLA(2)S. Specific tests are suggested to confirm the specificity of each assay. Additionally, it was discovered that one commonly used GIVA cPLA(2)/GVIA iPLA(2) inhibitor, methyl arachidonyl fluorophosphonate (MAFP) from one commercial source, was found to inhibit GIIA sPLA(2) and GV sPLA(2), but not GIVA cPLA(2), presumably due to oxidation of the compound during shipment, resulting in a different molecule with altered specificity. (c) 2005 Elsevier Inc. All rights reserved.