Purchase this article with an account.
T. M. Redmond, E. Poliakov, S. Gentleman; Modulation of Retinol Isomerase Activity in Visual Cycle-Expressing 293-F Cell Extracts. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5634.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
By utilizing site-directed mutagenesis and expression in mammalian cells of RPE65/retinol isomerase we hope to better understand the role of iron and its coordinating histidine residues in the catalytic mechanism of RPE65. This will involve perturbation of the iron center with iron specific agents. However supplementation of 293-F cells with chelators or iron causes cytotoxic death of the cells. Therefore, it was necessary to develop an in vitro assay using 293-F cell extracts.
293-F cells were transiently transfected with expression vectors for visual cycle proteins (RPE65, LRAT, CRALBP and RDH5). Membrane extracts of these cells were used to test the effect of additives on isomerase activity in vitro.
Firstly, we tested the effect of detergents on isomerase activity. Addition of CHAPS to 0.3% completely abolishes isomerase activity of cell extracts compared to no detergent control. Addition of dodecyl maltoside to 0.015%, in contrast, markedly enhances isomerase activity (about 2-fold) compared to no detergent control. Secondly, supplementation with exogenous ferrous iron was tested. Ferrous sulfate was added to 0, 1, 10 and 100 micromolar final concentration. Supplementation to 1 or 10 micromolar only slightly (10-15%) augmented isomerase activity, but supplementation to 100 micromolar slightly reduced isomerase activity, compared to the unsupplemented control. Thirdly, the effect of the ferrous chelator 2,2-dipyridyl (2,2-DP) on the assay system was tested. 250 micromolar 2,2-DP reduced isomerase activity to 15% of control. The IC50 of 2,2-DP was calculated to be 36 micromolar, tested over a range of 0-100 micromolar for this reagent.
We used membrane extracts of visual cycle expressing 293-F cell cells to measure RPE65/ retinol isomerase activity. Activity could be modulated by addition of key modifiers. Detergent choice is critical: CHAPS is deleterious to isomerase activity, whereas dodecyl maltoside enhances activity. Presumably, dodecyl maltoside preserves some critical native conformation or association not preserved by CHAPS. Addition of micromolar iron has slight effect on activity indicating that iron occupancy in expressed RPE65 molecules is almost total under the conditions studied. Conversely, RPE65/retinol isomerase activity is very sensitive to iron withdrawal, with a micromolar IC50 for 2,2-DP in this system. These data provide a baseline for further application of the system towards understanding RPE65/retinol isomerase catalysis.
This PDF is available to Subscribers Only