Figure 5.

Isomerase Activity in Sf9 Cells Expressing Rpe65 and Other Proteins

(A) 11cROL production in Sf9 cells infected with recombinant baculoviruses encoding the indicated proteins from atROL added to the culture medium.

(B) 11cROL production in Sf9 cells expressing the indicated proteins from atRP added to the medium.

(C) In vitro isomerase assay of membranes from Sf9 cells expressing the indicated proteins with atROL substrate. Levels of 11cROL are expressed in peak-area units (mAU) at 318 nm.

(D) In vitro isomerase assay of membranes from Sf9 cells expressing the indicated proteins with atRP substrate. Note the production of 11cROL from atRP by Sf9 cell membranes expressing only Rpe65.

(E) Representative chromatogram acquired at 318 nm from the in vitro isomerase assay of Sf9 cell membranes expressing Rpe65 with atRP substrate (D), scaled to show the 11cROL peak (arrow).

(F) UV spectrum acquired from the indicated 11cROL peak in (E).

(G) Representative chromatogram from the in vitro isomerase assay of membranes from Sf9 cells expressing GFP with atRP substrate (D).

(H) In vitro isomerase assay of membranes from Sf9 cells expressing Rpe65 with atRP substrate. Assay conditions were identical except for deletion or addition of 3 mM sodium cholate. Note the 9.2-fold higher isomerase activity in the presence of sodium cholate.

(I) Substrate saturation curves for 11cROL production from atRP by membranes from Sf9 cells expressing Rpe65 or GFP. Initial reaction rates (V₀) were determined at the indicated concentrations of atRP. The curve was fit by the Michaelis-Menten equation for a single-substrate reaction.

(J) Eadie-Hofstee transformation of the kinetic data in (I). The kinetic parameters V_max and K_M were derived from the x intercept and slope. Error bars show standard deviations (n = 4).