AN IMPROVED ASSAY FOR MEASURING THE TRANSVERSE REDISTRIBUTION OF FLUORESCENT PHOSPHOLIPIDS IN PLASMA-MEMBRANES

The internalization of fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl (N BD)-phospholipids from the plasma membrane can be assessed by the irre versible quenching of analogues in the outer leaflet by dithionite. He re we have utilized this assay to follow the redistribution of short-c hain C-6-NBD-sphingomyelin and C-6-NBD-phosphatidylserine from the cel l membrane of human gingiva fibroblasts. The significant uptake of dit hionite across the plasma membrane and the subsequent reduction of NBD -analogues exposed to the cytoplasmic lumen does not allow an accurate measurement of the amount of internalized lipid probes even at low te mperature. We could show that a precise determination can be achieved by extraction of analogues remaining in the exoplasmic half by a short pretreatment with bovine serum albumin prior to addition of dithionit e. The fluorescence of analogues localized to the cytoplasmic lumen wa s slowly destroyed by permeating dithionite. The fluorescence of those NBD-probes which are localized in the inner layer of intracellular ve sicles remained almost unaffected in the time course of the assay. Thu s, this approach allows to distinguish between different routes of int ernalization of NBD-phospholipids from the plasma membrane.