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 A2 (PLA2) types in biological samples. This approach should be useful in lipidomics studies. The assays were originally designed to differentiate between Group IVA cytosolic PLA2 (GIVA cPLA2), Group VIA calcium-independent PLA2 (GVIA iPLA2), Group IIA secreted PLA2 (GIIA sPLA2) and Group V secreted PLA2 (GV sPLA2). The specificity of these assays has now been confirmed using purified, recombinant human PLA2s 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 PLA2s. Specific tests are suggested to confirm the specificity of each assay. Additionally, it was discovered that one commonly used GIVA cPLA2/GVIA iPLA2 inhibitor, methyl arachidonyl fluorophosphonate (MAFP) from one commercial source, was found to inhibit GIIA sPLA2 and GV sPLA2, but not GIVA cPLA2, presumably due to oxidation of the compound during shipment, resulting in a different molecule with altered specificity.
Lucas
Karin Killermann
KK
University of California, San Diego School of Medicine, Department of Chemistry, Biochemistry and Pharmacology, 9500 Gilman Drive, La Jolla, San Diego, CA 92093 0601, USA.
Dennis
Edward A
EA
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D001671:Biochemistry / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054513:Group IV Phospholipases A2; D006801:Humans; D007249:Inflammation; D050356:Lipid Metabolism; D008823:Micelles; D063065:Organophosphonates / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013116:Spinal Cord / Q000378:metabolism
D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D008823:Micelles; D063065:Organophosphonates; D011994:Recombinant Proteins; C104810:methyl arachidonylfluorophosphonate; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; C516389:PLA2G4A protein, human; D054467:Phospholipases A2; C516391:Pla2g4a protein, rat; D010100:Oxygen