FIG. 3.
Characterization of reconstituted proteoliposomes. Proteoliposomes were reconstituted from mixtures of Triton X-100-solubilized egg PC and TE; liposomes were prepared similarly except that TE was omitted. (A) Silver-stained SDS-PAGE (12% acrylamide) gel of TE and reconstituted proteoliposomes (P). Molecular mass markers are indicated on the left. Samples of TE (30 μl) and proteoliposomes (prepared from 60 μl TE) were precipitated with trichloroacetic acid and washed with acetone before being dissolved in an SDS-containing sample buffer for PAGE analysis. (B) Collisional quenching of NBD fluorescence with iodide ions to determine the fraction of NBD-PL accessible on the outer leaflets of vesicles. Proteoliposomes (open squares; protein/phospholipid ratio, 4.5 mg/mmol) and liposomes (open circles) were reconstituted from Triton X-100-solubilized mixtures containing C12-NBD-PC. Asymmetrically labeled liposomes (filled circles) were prepared by adding C12-NBD-PC to preformed vesicles. The data are presented as modified Stern-Volmer plots (see Materials and Methods). Fo is the fluorescence intensity of the sample in the absence of quencher, whereas ΔF is the fluorescence intensity at a given iodide ion concentration. The connecting lines were obtained by linear regression. The inverse of the y-intercept represents the fraction of C12-NBD-PC that is accessible to the quencher.