UC San Diego

Phospholipase A₂ (PLA₂) constitutes a superfamily of enzymes that is well-studied by the scientific community. PLA₂s are membrane-associated enzymes that catalyze the hydrolysis of the ester bond at the sn-2 position of membrane phospholipids liberating free fatty acids, predominantly arachidonic acid (AA). The PLA2 superfamily consists of 16 groups and numerous subgroups and they can be considered as six major types: the secreted (sPLA₂), the cytosolic (cPLA₂), the calcium-independent (iPLA₂), the platelet-activating factor acetylhydrolase (PAF-AH), lysosomal PLA₂ (LPLA₂), and the adipose-PLA (AdPLA). The human Group IVA cPLA₂ (GIVA cPLA₂) was cloned and sequenced from U937 cells in 1999. This enzyme contains 749 amino acids and has a molecular weight of 85.2 kDa. The crystal structure of the enzyme was resolved in 1999 and was found to contain an N-terminal C2 domain and a C- terminal catalytic domain. GIVA cPLA₂ is of important biological relevance and involved at the earliest stage of inflammation since through its specificity for AA at the sn-2 position, it is the main AA provider for the eicosanoid biosynthetic pathway. Understanding the function of the enzyme is vital to find new ways to modulate inflammatory diseases in contrast to inhibiting traditional downstream pathways. In this study, we have designed a liquid chromatography/mass spectrometry (LC/MS) high-throughput assay to test the specificity of GIVA cPLA₂ toward different substrates. We tested various natural phospholipid substrates and found a preference for GIVA cPLA₂ for 1-palmitoyl-2-arachidonoyl-sn-glycero-3- phosphocholine (PAPC). We also validated the assay for determining inhibitors constants and potency using commercially available inhibitors