RAPID-DETERMINATION OF THE TRANSBILAYER DISTRIBUTION OF NBD-PHOSPHOLIPIDS IN ERYTHROCYTE-MEMBRANES WITH DITHIONITE

The assessment of the transverse distribution and mobility of NBD-labe Iled phospholipid analogues in biological membranes by selective chemi cal destruction of fluorescent label in the outer monolayer with dithi onite has been investigated using resealed erythrocyte ghosts as a mod el system. The distribution of those analogues can be determined in <3 0 s directly in the cell suspension provided the permeation of dithion ite across the membrane is suppressed. The results were compared with data on translocation of either NBD- or spin-labelled phospholipid ana logues obtained with the technique of back exchange to BSA. It is show n that the passage of dithionite can be mediated by anion-transport sy stems such as band 3 which is inhibited by DIDS. Appropriate condition s for the applicability of the assay were elucidated also using reseal ed ghosts having fluorescent NBD-taurine in the intracellular lumen. T he application of the assay to measure fast translocation processes, e .g. those mediated by the aminophospholipid translocase, is described.