Probing phospholipase a(2) with fluorescent phospholipid substrates.
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.
European Molecular Biology Laboratorium, Gene Expression Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Oliver
O
Wichmann
Michael H
MH
Gelb
Carsten
C
Schultz
D000818:Animals; D016718:Arachidonic Acid / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D001514:Bee Venoms / Q000201:enzymology; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology; D004735:Energy Transfer; D005227:Fatty Acids / Q000378:metabolism*; D005453:Fluorescence; D054501:Group II Phospholipases A2; D006367:HeLa Cells; D006801:Humans; D008049:Lipase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010179:Pancreas / Q000201:enzymology; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D013552:Swine
D001514:Bee Venoms; D005227:Fatty Acids; D010714:Phosphatidylethanolamines; D016718:Arachidonic Acid; D003300:Copper; D008049:Lipase; D010741:Phospholipases A; D054501:Group II Phospholipases A2