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Probing Phospholipase A 2 with Fluorescent Phospholipid Substrates
Author(s) -
Wichmann Oliver,
Gelb Michael H.,
Schultz Carsten
Publication year - 2007
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200600462
Subject(s) - arachidonic acid , chemistry , phospholipid , double bond , phosphatidylethanolamine , fluorescence , förster resonance energy transfer , fatty acid , phospholipase a , biochemistry , phospholipase a2 , stereochemistry , membrane , phosphatidylcholine , enzyme , organic chemistry , physics , quantum mechanics
The Foerster resonance energy transfer‐based sensor, PENN, measures intracellular phospholipase A 2 (PLA 2 ) activity in living cells and small organisms. In an attempt to modify the probe for the detection of particular isoforms, we altered the sn ‐2 fatty acid in such a way that either one or three of the Z double bonds in arachidonic acid were present in the sensor molecule. Arachidonic‐acid‐mimicking fatty acids were prepared by copper‐mediated coupling reactions. Probes with a single double bond in the 5‐position exhibited favorable substrate properties for secretory PLA 2 s. In vitro experiments with the novel unsaturated doubly labeled phosphatidylethanolamine derivatives showed preferred cleavage of the sensor PENN2 (one double bond) by the physiologically important group V sPLA 2 , while the O ‐methyl‐derivative PMNN2 was accepted best by the isoform from hog pancreas. For experiments in living cells, we demonstrated that bioactivation via S ‐acetylthioethyl (SATE) groups is essential for probe performance. Surprisingly, membrane‐permeant versions of the new sensors that contained double bonds, PENN2 and PENN3, were only cleaved to a minor extent in HeLa cells while the saturated form, PENN, was well accepted.