Determination of lipid asymmetry in human red cells by resonance energy transfer.
This report describes the application of a resonance energy transfer assay to determine the transbilayer distribution of 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled lipid analogues. The validity of this technique was established by determining the relationship between the distance of separation of lissamine rhodamine B labeled phosphatidylethanolamine (N-Rho-PE) acceptor lipid and NBD-labeled donor lipid and energy transfer efficiency. By determination of the distance between probes at 50% transfer efficiency (R0), the distance between fluorophores distributed symmetrically (outer leaflet label) and asymmetrically in artificially generated vesicles was determined. Calculation of the average distance between probes revealed a 14-A difference between NBD-lipid and N-Rho-PE localized in the same leaflet and in opposing leaflets, respectively. Application of this technique to the study of the transbilayer distribution of NBD-lipid in human red blood cells (RBC) showed that exogenously supplied NBD-phosphatidylserine (NBD-PS) was selectively transported to the inner leaflet, whereas NBD-phosphatidylcholine remained in the outer leaflet. In contrast, pretreatment of the RBC with diamide (a SH cross-linking reagent) blocked the transport of NBD-PS. The absence or presence of NBD-PS in the outer leaflet was independently verified by employing "back-exchange", trinitrobenzenesulfonic acid derivatization, and decarboxylation with PS decarboxylase experiments. These control experiments yielded results which confirmed the lipid distributions determined by the resonance energy transfer assay.
Connor
J
J
Department of Cell Biology, University of Texas M. D. Anderson Hospital and Tumor Institute at Houston 77030.
Schroit
A J
AJ
D009327:4-Chloro-7-nitrobenzofurazan; D004735:Energy Transfer; D004910:Erythrocyte Membrane / Q000648:ultrastructure*; D006801:Humans; D008051:Lipid Bilayers; D008563:Membrane Lipids / Q000097:blood*; D008954:Models, Biological; D010743:Phospholipids / Q000097:blood*; D013050:Spectrometry, Fluorescence
D008051:Lipid Bilayers; D008563:Membrane Lipids; D010743:Phospholipids; D009327:4-Chloro-7-nitrobenzofurazan