A Novel Assay for the Introduction of the Vinyl Ether Double Bond into Plasmalogens Using Pyrene-labeled Substrates

Overview

Journal J Lipid Res

Publisher Elsevier

Specialty Biochemistry

Date 2018 Mar 16

PMID 29540573

Citations 9

Authors

Ernst R Werner

Markus A Keller

Sabrina Sailer

Daniele Seppi

Georg Golderer

Gabriele Werner-Felmayer

Raphael A Zoeller

Katrin Watschinger

Affiliations

Soon will be listed here.

Abstract

Plasmanylethanolamine desaturase (PEDS) (EC 1.14.99.19) introduces the 1-prime double bond into plasmalogens, one of the most abundant phospholipids in the human body. This labile membrane enzyme has not been purified and its coding sequence is unknown. Previous assays for this enzyme used radiolabeled substrates followed by multistep processing. We describe here a straight-forward method for the quantification of PEDS in enzyme incubation mixtures using pyrene-labeled substrates and reversed-phase HPLC with fluorescence detection. After stopping the reaction with hydrochloric acid in acetonitrile, the mixture was directly injected into the HPLC system without the need of lipid extraction. The substrate, 1--pyrenedecyl-2-acyl--glycero-3-phosphoethanolamine, and the lyso-substrate, 1--pyrenedecyl--glycero-3-phosphoethanolamine, were prepared from RAW-12 cells deficient in PEDS activity and were compared for their performance in the assay. Plasmalogen levels in mouse tissues and in cultured cells did not correlate with PEDS levels, indicating that the desaturase might not be the rate limiting step for plasmalogen biosynthesis. Among selected mouse organs, the highest activities were found in kidney and in spleen. Incubation of intact cultivated mammalian cells with 1--pyrenedecyl--glycerol, extraction of lipids, and treatment with hydrochloric or acetic acid in acetonitrile allowed sensitive monitoring of PEDS activity in intact cells.

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