Journal of Lipid Research, Vol 28, 864-872, Copyright © 1987 by Lipid Research, Inc.
Synthesis of a naphthylvinyl-labeled glycerol ether analog of phosphatidylcholine and its use in the assay of phospholipase A2
HS Hendrickson, EK Hendrickson and TJ Rustad
The synthesis of a naphthylvinyl-labeled glycerol ether analog of
phosphatidylcholine, 1-O-[12-(2-naphthyl)-dodec-11-enyl]-2-O-decanoyl-
sn-glycerol-3- phosphocholine (NVPC), is described. This involves a Wittig
reaction between 2-naphthaldehyde and a phosphonium salt which gives the
trans-naphthylvinyl group as the predominant isomer. Lyso NVPC was prepared
from NVPC by phospholipase A2 action. NVPC absorbs strongly at 248 nm
(epsilon = 58,300 M-1 cm-1) and gives broad fluorescence emission with
maxima at 343 nm and 360 nm and a quantum yield of 0.10 in ethanol. An
assay for phospholipase A2 was developed using high performance liquid
chromatography with fluorescence detection to separate and quantify NVPC
and lyso NVPC. Activities as low as 1-2 pmol/min in an assay volume of 0.1
ml can easily be measured. The assay was used with a pure enzyme from cobra
venom and a crude enzyme from synovial fluid. Enzyme specificities for
phosphatidylcholine and NVPC with cobra venom and porcine pancreatic
phospholipases A2 were compared using a titrametric assay. The use of the
assay with NVPC to study the metabolism of platelet activating factor is
discussed.
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