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

In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) ha s been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radical s TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable o f oxidizing residues that contain primary hydroxyl and amino groups, When a pplied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipi d by thin-layer chromatography and that was identified by mass spectrometri c analysis as the corresponding acid glucuronylceramide, To test whether ox idation was confined to the external leaflet, TEMPO was applied to large un ilamellar vesicles (LUVs) consisting of egg phosphatidylcholine-egg phospha tidylethanolamine-cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiol abeled phosphatidylethanolamine, whereas carboxy -TEMPO oxidized only half. Hydrolysis by phospholipase Ap confirmed that 50% of the phosphatidylethan olamine was accessible in the external bilayer leaflet, suggesting that TEM PO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containing <1 mol% radiolabeled glucosylceramide or short-chain Cs-glu cosylceramide, carboxy-TEMPO oxidized half the glucosylceramide, However, i f surface Cs-glucosylceramide was first depleted by bovine serum albumin (B SA) (extracting 49 +/- 1%), 94% of the remaining C-6-glucosylceramide was r esistant to oxidation. Carboxy-TEMPO oxidized glucosylceramide on the surfa ce of LUVs without affecting inner leaflet glucosylceramide, At pH 9.5 and at 0 degrees C, the reaction reached completion by 20 min.