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Brandi Betts-Obregon, Andrew Mendiola, Andrew Tsin; Isomerase 2 Activity in Primary Müller Cells from cone-dominated Chicken Retinas is iron-dependent. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3767.
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Previous studies have shown that there are two parallel visual cycles in the cone dominated chicken retina (Muniz et al 2009). Takahashi et al 2011 showed that RPE65c is an iron-dependent Isomerase 2 in the retinal Müller cells of the cone dominant zebrafish. Kaylor et al 2012 showed that DES1 is an Isomerase 2 in the Müller glial cells of the retina. This DES1 protein has ion-binding di-iron ligands. We hypothesize that Müller cells located in the chicken retina have an iron-dependent Isomerase
Primary Müller cells were harvested from chicken retinas and seeded into T75 flasks. Cells were grown until confluence (approx 10-14 days) and homogenates prepared. Homogenates were pre-incubated with or without 5mM of 2,2’-Bipyridine (a known isomerase 2 inbhibitor) for 30 minutes at room temperature. Homogenates were then incubated with 20 µM all-trans retinol delivered in BSA along with 100 µM PalmCoA and 30 µM CRALBP for one hour in a 37C shaking water bath. Retinoids were extracted and analyzed by HPLC in comparison to retention times and authentic retinoid standards.
Bovine RPE homogenates (chelator control) yielded 80 pmol of 11cisRE/mg of protein when incubated with all-trans retinol. In the presence of 2,2’-bipyridne, the amount of 11-cisRE’s dropped to 38 pmol/mg of protein. Primary Chicken Müller cell homogenates incubated with all-trans retinol yielded 116 pmol of 11-cisRE/mg of protein. Primary Chicken Müller cell homogenates that were incubated with the iron chelator, 2,2’-bipyridine, yielded no 11-cisRE’s. Levels of 11-cis retinol remained constant at 24 pmol/mg of protein in homogenates incubated both with and without the addition of 2,2’-bypridine iron chelator.
Isomerase 2 activity in Müller cells of the cone-dominated chicken retina is iron-dependent.
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