Determination of lipid asymmetry in human red cells by resonance energy transfer

Journal Article · Tue Aug 11 04:00:00 EDT 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00390a031· OSTI ID:5722348

Connor, J; Schroit, A J

This report describes the application of a resonance energy transfer assay to determine the transbilayer distribution of /sup 125/I-labelled 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled 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 (R/sub 0/), 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 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.

• Research Organization: Univ. of Texas M.D. Anderson Hospital and Tumor Institute, Houston
• OSTI ID: 5722348
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:16; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550301* -- Cytology-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BETA DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CELL KILLING
CHROMIUM 51
CHROMIUM ISOTOPES
DAYS LIVING RADIOISOTOPES
DISTRIBUTION
ELECTRON CAPTURE RADIOISOTOPES
EMISSION SPECTRA
ERYTHROCYTES
EVEN-ODD NUCLEI
FLUORESCENCE
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
LIPIDS
LUMINESCENCE
MATERIALS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
RADIOISOTOPES
RESONANCE FLUORESCENCE
SPECTRA
SUBCELLULAR DISTRIBUTION
VIABILITY