DITHIONITE QUENCHING RATE MEASUREMENT OF THE INSIDE-OUTSIDE MEMBRANE BILAYER DISTRIBUTION OF 7-NITROBENZ-2-OXA-1,3-DIAZOL-4-YL-LABELED PHOSPHOLIPIDS

Measurement of the extent of dithionite quenching of the fluorescence of 7-nitrobenz-2-oxa-1,3-diazol-4-yl- (NBD-) labeled Lipids inserted i nto cellular and organellar membranes has been used to quantify their topological distribution and translocation. This assay provides a stra ightforward method for determining the fraction of NBD-Lipid exposed t o the outer leaflet of membranes that are impermeant to dithionite. Ho wever, it appears that many, if not all, cellular membranes are relati vely permeable to dithionite. The present work describes a method in w hich the initial rate of dithionite quenching, rather than the extent of quenching, was used to determine the fraction of different NBD-labe led phospholipids exposed to the outer leaflet. This method permits th e estimation of the translocation process even in experimental conditi ons where the membrane is semipermeable to dithionite. This technique was used to measure the translocation of several NBD-labeled phospholi pids across two biological membranes: brush border membranes vesicles (BBMV) from rabbit intestine and secretory vesicles (SV) from sec 6-4 mutant yeast cells. BBMV were shown to passively equilibrate diazol-4- yl)mono-palmitoylphosphatidylethanolamine (N-NBD-PPE) and a-1,3-diazol -4-yl)aminocaproyl]phosphatidylcholine (P-C-6-NBD-PC) to similar to 50 % in the inner leaflet by a protein-mediated process. In addition, P-C 6-NBD-PC was shown to passively equilibrate across SV to similar to 20 % in the inner leaflet, The addition of Mg2+ increased the amount on t he inner leaflet to similar to 30% by an unknown mechanism, but no evi dence for ATP-dependent inward translocation across the SV was found. In the case of BBMV, several different NBD-phospholipids were transloc ated from the outer to inner leaflet in a matter of minutes and reache d an equilibrium distribution of similar to 50% inside and outside. Th is movement was inhibitable by N-bromosuccinimide. The inward transloc ation rate and distribution of headgroup labeled N-NBD-lysophosphatidy lethanolamine, having one titratable negative charge, was increased in the presence of an inward basic pH gradient. The same NBD-phospholipi ds were also translocated across SV to roughly 50% in both leaflets wi th the exception of NBD-phosphatidic acid, which was passively distrib uted with 80% in the inner leaflet.