Abstract

Phospholipase A2 (PLA2) enzymes are a superfamily of lipolytic enzymes critical for cellular membrane homeostasis, signal transduction, and the initiation of inflammatory responses through the release of arachidonic acid. Given their central role in numerous physiological and pathological processes, including neurodegeneration, cardiovascular disease, and cancer, accurate and reliable methods for monitoring PLA2 activity are indispensable for both basic research and drug discovery. This review addresses the diverse methodologies employed for the quantitative assessment of PLA2 activity, focusing on techniques relevant to membrane and cell biology. We systematically evaluate established approaches, such as radiometric assays and spectrophotometric methods utilizing chromogenic or fluorogenic substrates, discussing their sensitivity, throughput, and applicability to different enzyme subclasses (e.g., secreted, cytosolic, and calcium-independent PLA2s). Furthermore, we explore advanced techniques, including mass spectrometry-based lipidomics and high-content screening platforms, which offer enhanced resolution and the ability to monitor activity in complex cellular environments. The objective is to provide a comprehensive guide to the advantages and limitations of current PLA2 activity monitoring methods, thereby facilitating the selection of appropriate assays for studying enzyme kinetics, inhibitor efficacy, and the functional role of PLA2 in membrane dynamics and cellular signaling pathways.