Analyses of Calcium-Independent Phospholipase A₂beta (iPLA₂β) in Biological Systems

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.