Measurement of phospholipase A2 and 1-alkylglycerophosphocholine acetyltransferase activities in stimulated alveolar macrophages by HPLC analysis of NBD-labeled ether lipids.
The importance of phospholipases in cellular signaling and 1-alkylglycerophosphocholine acetyltransferase in the formation of platelet-activating factor (PAF) has stimulated demand for methods to measure these enzyme activities in inflammatory cells. Most of the assays currently used rely on radiolabeled substrates. We have synthesized NBD-labeled ether lipids as substrates for measuring enzyme activities of the PAF cycle and of lysosomal phospholipase A2 (PLA2). The fluorescent lipids were incubated with homogenates of stimulated bovine alveolar macrophages. The generated products were separated from the substrates by HPLC on a normal phase and monitored with a fluorescence detector. NBD-lyso-PAF was well accepted by acetyl- and acyltransferases of the cell-free preparations, which metabolized the substrate into NBD-PAF and NBD-alkyl-acylglycerophosphocholines. Homogenates of stimulated cells showed an enhanced production of NBD-PAF. The increased formation of the biological mediator was dependent on the nature of the stimuli and the time of stimulation. Lysosomal PLA2 was measured with 1-O-(12-NBD-aminododecyl)-2-acyl-sn-glycero-3-phosphocholine as substrate. By varying the pH and the calcium concentration, it was possible to distinguish between the cytosolic PLA2 and the lysosomal PLA2 activity. Optimal conditions for the determination of the lysosomal PLA2 were obtained at pH 4.5 and in the presence of EDTA. Stimulation with particulate agonists induced an enhancement of the lysosomal PLA2 activity in macrophages.
Institut fur Biochemie und Lebensmittelchemie, Abteihung fur Biochemie und Molekularbiologie, Universitat Hamburg, Germany.
B
B
Kleuser
A
A
Meister
L
L
Sternfeld
G
G
Gercken
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000123:Acetyltransferases / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007476:Ionophores / Q000494:pharmacology; D008247:Lysosomes / Q000201:enzymology; D016676:Macrophages, Alveolar / Q000201:enzymology*; D010587:Phagocytosis / Q000187:drug effects; D010741:Phospholipases A / Q000032:analysis / Q000378:metabolism*; D054467:Phospholipases A2; D010742:Phospholipid Ethers / Q000032:analysis* / Q000378:metabolism; D010972:Platelet Activating Factor / Q000378:metabolism; D012822:Silicon Dioxide / Q000494:pharmacology; D017968:Vanadium Compounds / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007476:Ionophores; D010742:Phospholipid Ethers; D010972:Platelet Activating Factor; D017968:Vanadium Compounds; D012822:Silicon Dioxide; D015054:Zymosan; D000123:Acetyltransferases; C046035:1-alkylglycerophosphocholine acetyltransferase; D010741:Phospholipases A; D054467:Phospholipases A2; D009327:4-Chloro-7-nitrobenzofurazan