Fig. 3.

Dependence of isomerohydrolase activity on RPE65 expression levels. (A) Time course of 11-cis retinol generation in 293A cell lysates expressing both LRAT and RPE65. (B) Time course of 11-cis retinol generation in bovine RPE homogenates. (C) Dependence of isomerohydrolase activity on MOI of Ad-RPE65. Lysates (250 μg) of 293A cells infected by Ad-RPE65 with increasing MOI and transfected with the same amount of the LRAT plasmid or 33 μg of bovine RPE homogenate were incubated with 0.2 μM all-trans [³H]retinol. In A–C, 11-cis [³H]retinol generated from the reaction were calculated from the area of the 11-cis retinol peak (mean ± SD, n = 3). (D) Western blot analysis of the same lysates used for the activity assay in C. PC, positive control, 10 μg of microsomal proteins from bovine RPE (for the RPE65 blot) and 1.25 μg of cell lysates of Sf9 cell expressing LRAT (for the LRAT blot); NC, negative control (40 μg of total protein from untreated cells); remaining lanes: 40 μg cell lysates from cells transfected with the same amount of LRAT plasmid and infected with Ad-RPE65 at different MOI as indicated. (E) Dependence of isomerohydrolase activity on the RPE65 expression level.