Purchase this article with an account.
K. M. Farjo, G. Moiseyev, Y. Takahashi, J.-X. Ma; Retinol Dehydrogenase 10 is a Potential 11-cis-RDH in the RPE Retinoid Visual Cycle. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3398.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinol dehydrogenase 10 (RDH10) was originally identified from the retinal pigment epithelium (RPE) and Müller cells. The goal of this study is to characterize the 11-cis-retinol dehydrogenase (11-cis-RDH) activity of RDH10 in vitro and in a cell culture model. We specifically sought to determine: 1) how cellular retinaldehyde binding protein (CRALBP) affects RDH10 activity and 2) if RDH10 can cooperate with RPE protein 65kDa (RPE65) in cell culture to reconstitute the RPE retinoid cycle.
To evaluate the 11-cis-RDH activity of RDH10 in vitro, COS1 cells were transfected with an RDH10 expression vector and cellular membrane fractions were isolated 2 days post-transfection. Membrane fractions were combined with NAD+ or NADP+ and 11-cis-retinol substrate in the presence or absence of increasing molar ratios of CRALBP/11-cis-retinol. Following incubation, retinoids were extracted and analyzed by HPLC. The 11-cis-RDH activity of RDH10 was tested in cell culture by co-transfecting RDH10, CRALBP, and RPE65 expression vectors into HEK-293A cells stably expressing lecithin retinol acyltransferase (LRAT). RDH5 and RDH8 were transfected in place of RDH10 as positive and negative controls, respectively. Cells were treated with all-trans-retinoland collected for retinoid extraction and analysis by HPLC.
In vitro studies confirm that RDH10 has 11-cis-RDH activity and may prefer the NAD+ cofactor (Km = 3 µM, Vmax = 6.12 nmol/mg/hr). Furthermore, RDH10 can oxidize 11-cis-retinol in the presence of CRALBP. In a cell culture model that reconstitutes the RPE visual cycle, all-trans-retinol was sequentially converted to 11-cis-retinol by LRAT and RPE65 and then to the visual chromophore 11-cis-retinal by RDH10.
RDH10 has 11-cis-RDH activity in vitro and can act in concert with other enzymes of the RPE retinoid cycle in cell culture. CRALBP does not significantly inhibit RDH10 activity, suggesting RDH10 has the ability to use CRALBP-bound 11-cis-retinol as substrate, and thus could fulfill the role of 11-cis-RDH, together with RDH5, in the RPE retinoid visual cycle in vivo.
This PDF is available to Subscribers Only