Development and Application of Novel Methodologies for the Characterization of Phospholipase A2 Activity: Targeted Lipidomic Profiling and Interfacial Binding Analysis
Bollinger, James Gerard
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Phospholipases A2 are a family of enzymes that hydrolyze membrane glycerophospholipids to yield free fatty acids and lysophospholipids. Both products serve as precursors for an array of lipid mediators that play known roles in a variety of signal transduction processes. Discussed herein is the development and application of novel analytical methods to characterize both the kinetic and interfacial binding parameters of members of the secreted and cytosolic PLA2 families. Specifically, the discussion involves the development of assays that utilize the triple quadrupole LC-ESI-MS/MS analytical platform to profile the products and relevant downstream metabolites for the PLA2 enzymatic reaction; lysophospholipids, free fatty acids, and eicosanoids, a family of bioactive lipid mediators derived from arachidonic acid. Additionally, the discussion includes methods to characterize the interfacial binding behavior for several prominent members of the secreted PLA2 family as well as their associated point mutants.
- Chemistry