FIGURE 3.

Characterization of Rho proteoliposomes. A, schematic representation of Rho orientation within proteoliposomes. B, functional characterization of incorporated Rho. Spectrophotometric analyses of changes in absorption spectra observed after proteoliposomes were exposed to light indicate Meta II state formation. The difference spectrum was calculated by subtracting the spectrum recorded after and before light exposure. AU, absorbance units. C, determination of Rho molecular orientation by proteolytic digestion with Asp-N endoprotease, which specifically cleaves the C terminus of Rho. Lanes 1 and 3 represent washed intact proteoliposomes prepared in the presence or absence of NH₂OH. Lanes 2 and 4 (protease-treated samples) reveal dominant protein bands corresponding to the Rho proteolytic fragment. D, densitometric quantification of protein bands shown in C indicate that at least 85% of Rho molecules preferentially adopt an orientation in which the N termini face the lumen of proteoliposomes.