Rapid determination of the transbilayer distribution of NBD-phospholipids in erythrocyte membranes with dithionite.
The assessment of the transverse distribution and mobility of NBD-labelled phospholipid analogues in biological membranes by selective chemical destruction of fluorescent label in the outer monolayer with dithionite has been investigated using resealed erythrocyte ghosts as a model system. The distribution of those analogues can be determined in < 30 s directly in the cell suspension provided the permeation of dithionite across the membrane is suppressed. The results were compared with data on translocation of either NBD- or spin-labelled phospholipid analogues obtained with the technique of back exchange to BSA. It is shown that the passage of dithionite can be mediated by anion-transport systems such as band 3 which is inhibited by DIDS. Appropriate conditions for the applicability of the assay were elucidated also using resealed ghosts having fluorescent NBD-taurine in the intracellular lumen. The application of the assay to measure fast translocation processes, e.g. those mediated by the aminophospholipid translocase, is described.
Humboldt-Universität zu Berlin, Institut für Biophysik, Germany.
T
T
Pomorski
A
A
Herrmann
A
A
Zachowski
P F
PF
Devaux
P
P
Müller
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D004227:Dithionite; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D010069:Oxadiazoles; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013654:Taurine / Q000031:analogs & derivatives
C078632:1-acyl-2-(12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D008051:Lipid Bilayers; C027027:NBD-taurine; D010069:Oxadiazoles; D010713:Phosphatidylcholines; D010743:Phospholipids; D013113:Spin Labels; D004227:Dithionite; D013654:Taurine; D009327:4-Chloro-7-nitrobenzofurazan