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Substrate-Specific Inhibition Constants for Phospholipase A<sub>2</sub> Acting on Unique Phospholipid Substrates in Mixed Micelles and Membranes Using Lipidomics
Abstract
Assaying lipolytic enzymes is extremely challenging because they act on water-insoluble lipid substrates, which are normally components of micelles, vesicles, and cellular membranes. We extended a new lipidomics-based liquid chromatographic–mass spectrometric assay for phospholipases A2 to perform inhibition analysis using a variety of commercially available synthetic and natural phospholipids as substrates. Potent and selective inhibitors of three recombinant human enzymes, including cytosolic, calcium-independent, and secreted phospholipases A2 were used to establish and validate this assay. This is a novel use of dose–response curves with a mixture of phospholipid substrates, not previously feasible using traditional radioactive assays. The new application of lipidomics to developing assays for lipolytic enzymes revolutionizes in vitro testing for the discovery of potent and selective inhibitors using mixtures of membranelike substrates- Text
- Journal contribution
- Biochemistry
- Pharmacology
- Biotechnology
- Hematology
- Infectious Diseases
- Computational Biology
- Chemical Sciences not elsewhere classified
- novel use
- Unique Phospholipid Substrates
- inhibition analysis
- assay
- Lipidomics Assaying lipolytic enzymes
- phospholipase
- 2 Acting
- Mixed Micelles
- mixture
- lipolytic enzymes revolutionizes
- phospholipid substrates
- Substrate-Specific Inhibition Constants
- membranelike substrates
- water-insoluble lipid substrates
- inhibitor