title: Analyses of Calcium-Independent Phospholipase A2beta (iPLA2β) in Biological Systems.
abstract: The Ca2+-independent phospholipases A2(iPLA2s) are part of a diverse family of PLA2s, manifest activity in the absence of Ca2+, are ubiquitous, and participate in a variety of biological processes. Among the iPLA2s, the cytosolic iPLA2β has received considerable attention and ongoing studies from various laboratories suggest that dysregulation of iPLA2β can have a profound impact on the onset and/or progression of many diseases (e.g., cardiovascular, neurological, metabolic, autoimmune). Therefore, appropriate approaches are warranted to gain a better understanding of the role of iPLA2β in vivo and its contribution to pathophysiology. Given that iPLA2β is very labile, its basal expression is low in a number of cell systems, and that crystal structure of iPLA2β is not yet available, careful and efficient protocols are needed to appropriately assess iPLA2β biochemistry, dynamics, and membrane association. Here, step-by-step details are provided to (a) measure iPLA2β-specific activity in cell lines or tissue preparations (using a simple radiolabel-based assay) and assess the impact of stimuli and inhibitors on resting- and disease-state iPLA2β activity, (b) purify the iPLA2β to near homogeneity (via sequential chromatography) from cell line or tissue preparations, enabling concentration of the enzyme for subsequent analyses (e.g., proteomics), and (c) employ hydrogen/deuterium exchange mass spectrometry analyses to probe both the structure of iPLA2β and dynamics of its association with the membranes, substrates, and inhibitors.
authors.affiliation: University of Georgia at Athens, Athens, GA, United States.
authors.forename: S E
authors.initials: SE
authors.lastname: Barbour
authors.affiliation: Comprehensive Diabetes Center, University of Alabama at Birmingham, Birmingham, AL, United States. Electronic address: sramvem@uab.edu.
authors.forename: S
authors.initials: S
authors.lastname: Ramanadham
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism; D002250:Carbon Radioisotopes; D002460:Cell Line; D041961:Deuterium Exchange Measurement; D057075:Enzyme Assays; D054522:Group VI Phospholipases A2 / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007659:Ketones / Q000494:pharmacology; D051379:Mice; D018482:Muscle, Skeletal / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry; D063065:Organophosphonates / Q000494:pharmacology; D010742:Phospholipid Ethers / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: C400184:1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine; D001095:Arachidonic Acids; D002250:Carbon Radioisotopes; D007659:Ketones; D063065:Organophosphonates; D010742:Phospholipid Ethers; C104810:methyl arachidonylfluorophosphonate; C109341:palmitoyl trifluoromethyl ketone; C081565:arachidonyltrifluoromethane; C043051:1-O-hexadecyl-2-arachidonyl-sn-glycero-3-phosphocholine; D054522:Group VI Phospholipases A2; C516475:PLA2G6 protein, human; C516476:Pla2g6 protein, mouse; D002118:Calcium
keywords: Chromatographic purification; Hydrogen/deuterium exchange; In vitro activity assay; Phospholipase A(2); iPLA(2)β