Analyses of Calcium-Independent Phospholipase A 2 beta (iPLA 2 β) in Biological Systems.

The Ca2+ -independent phospholipases A2 (iPLA2 s) are part of a diverse family of PLA2 s, manifest activity in the absence of Ca2+ , are ubiquitous, and participate in a variety of biological processes. Among the iPLA2 s, the cytosolic iPLA2 β has received considerable attention and ongoing studies from various laboratories suggest that dysregulation of iPLA2 β 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 iPLA2 β in vivo and its contribution to pathophysiology. Given that iPLA2 β is very labile, its basal expression is low in a number of cell systems, and that crystal structure of iPLA2 β is not yet available, careful and efficient protocols are needed to appropriately assess iPLA2 β biochemistry, dynamics, and membrane association. Here, step-by-step details are provided to (a) measure iPLA2 β-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 iPLA2 β activity, (b) purify the iPLA2 β 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 iPLA2 β and dynamics of its association with the membranes, substrates, and inhibitors.