Abstract
We purified retinol dehydrogenase from bovine rod outer segments using polyethylene glycol precipitation and hydroxylapatite, concanavalin A-Sepharose CL-4B, and Sepharose CL-6B column chromatography in the presence of NADP. We obtained 13-fold purification of retinol dehydrogenase with specific activity of 61.8 nmol/min/mg and 3.8% recovery. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that retinol dehydrogenase had a molecular mass of 37,000 daltons. The Km values of purified retinol dehydrogenase for all-trans retinol and all-trans retinal were 6.6 mM and 0.085 mM, respectively. The purified enzyme reacted with the all-trans retinal but not with 13-, 11-, and 9-cis compounds. In addition, we prepared antibody to retinol dehydrogenase using rat. The anti-retinol dehydrogenase antibody precipitated retinol dehydrogenase activity and was confirmed to bind to 37-kDa protein by Western blotting. We also found that anti-retinol dehydrogenase antibody bound to bovine rod outer segments specifically by immunohistochemical technique. The molar ratio of retinol dehydrogenase to opsin in rod outer segments estimated by enzyme-linked immunosorbent assay was 1:140.
Highlights
We purified retinol dehydrogenase from bovine rod outer segments using polyethylene glycol precipitation and hydroxylapatite, concanavalin A-Sepharose CL
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that retinol dehydrogenase had a molecular mass of 37,000 daltons
All-trans retinal from bleaching rhodopsin is converted to all-trans retinobyl all-trans-specific retinol dehydrogenase in the rod outer segments of photoreceptor cells [1].All-trans retinol derived from the bleaching photoreceptor cells [2], andthe choroidal blood supply [3] is delivered to the retinal pigment epithelial (RPE)’ cells and is converted to 11-cis retinal through esterification [4,5,6], deesterification [7], isomerization (8-lo), and oxidation by 11cis-specific retinol dehydrogenase [1, 11].The 11-cis retinal formed in the RPE cells is delivered to rod outer segments (ROS) to regenerate rhodopsin [2, 12,13,14]
Summary
From the Department of Ophthalmology, TohokuUniuersity School of Medicine, 1-1 Seiryo-machi, Sendai, Miyagi 980, Japan. We purified retinol dehydrogenase from bovine rod outer segments using polyethylene glycol precipitation and hydroxylapatite, concanavalin A-Sepharose CL-. The K,,, values of purified retinol dehydrogenase for all-trans retinol and all-trans retinal were 6.6 mM and 0.085 mM, respectively. We found that anti-retinol dehydrogenase antibody bound to bovine rod outer segments by immunohistochemical technique. The molar ratio of retinol dehydrogenase to opsin in rod outer segments estimated by enzyme-linked immunosorbent assay was 1:140. The abbreviations used are: RPE, retinal pigment epithelial; ROS, rod outer segment; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. We found that retinol dehydrogenase, even solubilized with detergents, is not so labile in the presence of NADP. We purified the enzyme from bovine ROS and partially characterized it
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