FIG. 4.
Reconstitution of phospholipid flippase activity in proteoliposomes. Vesicles were prepared from Triton X-100-solubilized egg PC, TE, and either 1-C14-2-C6-NBD-PC, 1-acyl-2-C6-NBD-PC, 1-acyl-2-C12-NBD-PC, or PE. Different amounts of TE were used to generate vesicle populations with different PPR (expressed in milligrams per millimole). (A) Plot of the extent of BSA-extractable 1-C14-2-C6-NBD-PC as a function of the PPR. (B) Plot showing the percentage of reduction in fluorescence obtained upon addition of dithionite to the reconstituted vesicle preparations. The data are fitted by a monexponential function that is simplified by deconvolution into two linear segments: a line of positive slope generated by linear regression of data points from the rising section of the graph and a plateau determined by the monoexponential curve fit. The point where the rising linear segment intersects the plateau (PPR, ∼10 mg/mmol) is interpreted as the point where each vesicle contains a single functional flippase. The fatty acid composition at the 1 position of the acyl-NBD-PLs used in panel B is as follows: palmitic acid, 62%; stearic acid, 29%; oleic acid, 5.5%.