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Journal of Lipid Research, Vol. 41, 1252-1260, August 2000
Copyright © 2000 by Lipid Research, Inc.

Original Article

Assay for the transbilayer distribution of glycolipids: selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

Correspondence to: Gerrit van Meer

In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) has been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radicals TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable of oxidizing residues that contain primary hydroxyl and amino groups. When applied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipid by thin-layer chromatography, and that was identified by mass spectrometric analysis as the corresponding acid glucuronylceramide. To test whether oxidation was confined to the external leaflet, TEMPO was applied to large unilamellar vesicles (LUVs) consisting of egg phosphatidylcholine– egg phosphatidylethanolamine;–cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiolabeled phosphatidylethanolamine, whereas carboxy-TEMPO oxidized only half. Hydrolysis by phospholipase A₂ confirmed that 50% of the phosphatidylethanolamine was accessible in the external bilayer leaflet, suggesting that TEMPO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containing <1 mol% radiolabeled glucosylceramide or short-chain C₆-glucosylceramide, carboxy-TEMPO oxidized half the glucosylceramide. However, if surface C₆-glucosylceramide was first depleted by bovine serum albumin (BSA) (extracting 49 ± 1%), 94% of the remaining C₆-glucosylceramide was resistant to oxidation. Carboxy-TEMPO oxidized glucosylceramide on the surface of LUVs without affecting inner leaflet glucosylceramide. At pH 9.5 and at 0°C, the reaction reached completion by 20 min.—Sillence, D. J., R. J. Raggers, D. C. A. Neville, D. J. Harvey, and G. van Meer. Assay for the transbilayer distribution of glycolipids: selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals. J. Lipid Res. 2000. 41: 1252;–1260.

Supplementary key words: glycosphingolipid, lipid asymmetry, ABC transporters

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