Purification of retinol dehydrogenase from bovine retinal rod outer segments.
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.
Ishiguro
S
S
Department of Ophthalmology, Tohoku University School of Medicine, Miyagi, Japan.
Suzuki
Y
Y
Tamai
M
M
Mizuno
K
K
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification*; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002417:Cattle; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D011233:Precipitin Tests; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase