US EPA

6791498 

Journal Article 

Measurement of phospholipase A(2) and 1-alkylglycerophosphocholine acetyltransferase activities in stimulated alveolar macrophages by HPLC analysis of NBD-labeled ether lipids 

Kleuser, B; Meister, A; Sternfeld, L; Gercken, G; , 

1996 

Yes 

Chemistry and Physics of Lipids
ISSN: 0009-3084 

ELSEVIER SCI IRELAND LTD 

CLARE 

29-37 

8907240 

WOS:A1996TW79300004 

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 A(2) (PLA(2)). 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 PLA(2) was measured with 1-0-(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 PLA(2) and the lysosomal PLA(2) activity, Optimal conditions for the determination of the lysosomal PLA(2) were obtained at pH 4.5 and in the presence of EDTA. Stimulation with particulate agonists induced an enhancement of the lysosomal PLA(2) activity in macrophages.