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 into cellular and organellar membranes has been used to quantify their topological distribution and translocation. This assay provides a straightforward method for determining the fraction of NBD-Lipid exposed to the outer leaflet of membranes that are impermeant to dithionite. However, it appears that many, if not all, cellular membranes are relatively permeable to dithionite. The present work describes a method in which the initial rate of dithionite quenching, rather than the extent of quenching, was used to determine the fraction of different NBD-labeled phospholipids exposed to the outer leaflet. This method permits the estimation of the translocation process even in experimental conditions where the membrane is semipermeable to dithionite. This technique was used to measure the translocation of several NBD-labeled phospholipids 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 N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)mono-palmitoylphosphatidylethanolamine (N-NBD-PPE) and 1-palmitoyl-2-[6-(7-nitrobenz-2-oxa-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-C6-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 the inner leaflet to similar to 30% by an unknown mechanism, but no evidence for ATP-dependent inward translocation across the SV was found. In the case of BBMV, several different NBD-phospholipids were translocated from the outer to inner leaflet in a matter of minutes and reached an equilibrium distribution of similar to 50% inside and outside. This movement was inhibitable by N-bromosuccinimide. The inward translocation rate and distribution of headgroup labeled N-NBD-lysophosphatidylethanolamine, having one titratable negative charge, was increased in the presence of an inward basic pH gradient. The same NBD-phospholipids were also translocated across SV to roughly 50% in both leaflets with the exception of NBD-phosphatidic acid, which was passively distributed with 80% in the inner leaflet.