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. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: FEBS J. 2011 Feb 3;278(6):973–987. doi: 10.1111/j.1742-4658.2011.08019.x

Figure 3. Zebrafish 13cIMH is a 13-cis specific isomerohydrolase.

Figure 3

The expression plasmids of human RPE65, zebrafish 13cIMH and red fluorescent protein (RFP, negative control) were separately transfected into 293A-LRAT cells. (a) Protein expression was confirmed by Western blot analyses. (b-d) Equal amounts of total cellular proteins from the cells (125 μg) expressing RFP (b), human RPE65 (c) and 13cIMH (d) were incubated with liposomes containing atRE (250 μM lipids, 3.3 μM atRE) for 1 hour at 37°C, and the generated retinoids were analyzed by HPLC. (e-f) A peak 3 in panel (d) was identified as the generated 13cROL based on retention time (d) and the absorption spectrum (e) compared to retention time (f) and absorption spectrum (inset) of the 13cROL standard. The x-axis of inset in panel (f) represents wavelength (nm). (g, h) For further confirmation of the identity of generated 13cROL, the 13cROL standard was spiked into the reaction products. The 13cROL peaks before (g) and after (h) the spike. The peaks were identified as follows: 1, retinyl esters; 2, 11cROL; 3, 13cROL.