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Substrate-Specific Inhibition Constants for Phospholipase A(2) Acting on Unique Phospholipid Substrates in Mixed Micelles and Membranes Using Lipidomics

[Image: see text] 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 phospholipas...

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Autores principales: Mouchlis, Varnavas D., Armando, Aaron, Dennis, Edward A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2019
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398150/
https://www.ncbi.nlm.nih.gov/pubmed/30615445
http://dx.doi.org/10.1021/acs.jmedchem.8b01568
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author Mouchlis, Varnavas D.
Armando, Aaron
Dennis, Edward A.
author_facet Mouchlis, Varnavas D.
Armando, Aaron
Dennis, Edward A.
author_sort Mouchlis, Varnavas D.
collection PubMed
description [Image: see text] 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 A(2) 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 A(2) 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.
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spelling pubmed-63981502019-03-05 Substrate-Specific Inhibition Constants for Phospholipase A(2) Acting on Unique Phospholipid Substrates in Mixed Micelles and Membranes Using Lipidomics Mouchlis, Varnavas D. Armando, Aaron Dennis, Edward A. J Med Chem [Image: see text] 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 A(2) 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 A(2) 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. American Chemical Society 2019-01-07 2019-02-28 /pmc/articles/PMC6398150/ /pubmed/30615445 http://dx.doi.org/10.1021/acs.jmedchem.8b01568 Text en Copyright © 2019 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
spellingShingle Mouchlis, Varnavas D.
Armando, Aaron
Dennis, Edward A.
Substrate-Specific Inhibition Constants for Phospholipase A(2) Acting on Unique Phospholipid Substrates in Mixed Micelles and Membranes Using Lipidomics
title Substrate-Specific Inhibition Constants for Phospholipase A(2) Acting on Unique Phospholipid Substrates in Mixed Micelles and Membranes Using Lipidomics
title_full Substrate-Specific Inhibition Constants for Phospholipase A(2) Acting on Unique Phospholipid Substrates in Mixed Micelles and Membranes Using Lipidomics
title_fullStr Substrate-Specific Inhibition Constants for Phospholipase A(2) Acting on Unique Phospholipid Substrates in Mixed Micelles and Membranes Using Lipidomics
title_full_unstemmed Substrate-Specific Inhibition Constants for Phospholipase A(2) Acting on Unique Phospholipid Substrates in Mixed Micelles and Membranes Using Lipidomics
title_short Substrate-Specific Inhibition Constants for Phospholipase A(2) Acting on Unique Phospholipid Substrates in Mixed Micelles and Membranes Using Lipidomics
title_sort substrate-specific inhibition constants for phospholipase a(2) acting on unique phospholipid substrates in mixed micelles and membranes using lipidomics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398150/
https://www.ncbi.nlm.nih.gov/pubmed/30615445
http://dx.doi.org/10.1021/acs.jmedchem.8b01568
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