FIGURE 2.

Cell-free synthesis of active TbSLS4. TbSLS4 was synthesized in the cell-free system, and proteoliposomes were recovered by floatation and assayed for conversion of NBD-ceramide substrate. A, samples from the gradient were fractionated by SDS-PAGE and stained with Coomassie Blue. Gradient fractions are indicated from top (T) to bottom (B), and the direction of floatation is indicated (arrow). A star indicates a TbSLS4-specific band recovered in the proteoliposomes. This band is absent in floated fraction 2 from reactions programmed for synthesis of bacteriorhodopsin (BO, indicated by arrowhead). B, recovered TbSLS4 or bacteriorhodopsin proteoliposomes were used for NBD-SL synthesis over a 4-h time course in MBS (pH 6.5, 0.25 μm NBD-ceramide, 30 °C); products were visualized by TLC and phosphorimaging. Mobilities of NBD standards, solvent front (F), NBD-ceramide (C), and origin (O) are indicated.