FIGURE 6.

Impacts of the site-directed mutations and fragment replacement with those of cRPE65 on protein levels and enzymatic activities of hRPE65. The identified site-directed mutants in this study (N170K and K297G) and the F1 chimera were combined to produce sIMH (F1/N170K/K297G). The identified point mutants and the F1 chimera of RPE65 were expressed in 293A-LRAT cells. The cells were harvested 48 h after the transfection, and protein levels of RPE65 were confirmed by Western blot analyses (A). Enzymatic activities were measured by the in vitro isomerohydrolase assay and quantified by generated 11-cis-[³H]retinol (B). Isomerohydrolase activities were quantified by generated 11-cis-[³H]retinol (pmol/h) and presented as a percentage of WT hRPE65 activity (mean ± S.E., n = 3).