FIG. 5. Isomeric form of the substrate of mouse RetSat.
Tet-induced HEKK-RetSat cells were incubated overnight with pure isomers of retinol (>95% pure by HPLC, assayed before incubation). Following incubation, retinoids were extracted and analyzed by normal phase HPLC. The appearance of 13,14-dihydroretinol isomers was monitored at 290 nm, since the absorbance maxima of most isomers of 13,14-dihydroretinol differ by less than 5 nm from 290 nm, the λmax of all-trans-13,14-dihydroretinol (spectra not shown). In each panel an arrow indicates the substrate investigated to distinguish it from the additional retinol isomers that were generated by thermal isomerization during overnight incubation in tissue culture. The numbers indicate the identity of eluted peaks based on absorbance spectra and comparison with pure standards, specifically 13-cis-retinol (1), all-trans-13,14-dihydroretinol (2), 9-cis-retinol (3), all-trans-retinol (4), 9,13-di-cis-retinol (5), and 11-cis-retinol (6). No isomers of 13,14-dihydroretinol were detected other than the all-trans isomer. The retention times in the bottom right panel are slightly longer due to variations in the solvent system. The experiment was performed in triplicate and repeated.