Measurement of NBD-PS Translocase Activity in Reconstituted Yeast Microsomal Vesicles.
The assay was based on the reduction of the fluorescent aryl-nitro group of NBD phospholipids into a nonfluorescent aryl-amino group. Fluorescence intensity was recorded for 10 min (F
T). The first arrow indicates addition of 30 μL of 1 M sodium dithionite in 1 M Tris-HCl, pH 10.0, to the reconstituted microsomes; the fluorescence was recorded after 10 min (F
D). Membrane-impermeable sodium dithionite reduces the fluorescent aryl-nitro group of NBD phospholipids present in the outer leaflet of the vesicle membrane (Suzuki et al., 1997). The second arrow indicates addition of 100 μL of 30% Triton X-100 to disrupt the microsomal vesicles and allow sodium dithionite to quench fluorescence from the inner leaflet–resident NDB phospholipids; the fluorescence was recorded (F
0). The fractions of NBD phospholipids present in the inner and outer leaflets were calculated from the following equations (Suzuki et al., 1997):
;
. −ATP, ATP was omitted from the assay; +ATP, ATP (3 mM) was included in the reaction mixture; AU, arbitrary units.