IDENTIFICATION AND IMMUNOHISTOCHEMISTRY OF RETINOL DEHYDROGENASE FROM BOVINE RETINAL-PIGMENT EPITHELIUM

We studied the properties of retinol dehydrogenase (11-cis-specific) from bovine retinal pigment epithelium. Detergents caused a loss of retinol dehydrogenase activity; therefore, we added 3 mM NADH as a stabilizer to solubilize this enzyme and partially purified this enzyme using Sepharose CL-6B and hydroxyapatite column chromatography. The partially-purified sample, which contained two major proteins (66 kDa, 33 kDa), had substrate preference to 11-cis and 13-cis-retinal but not to all-trans and 9-cis isomers. Monoclonal anti-33 kDa protein antibody precipitated oxidation and reduction activity of retinol dehydrogenase, and was confirmed to bind specifically to 33 kDa protein of retinal pigment epithelial crude extract by Western blotting. In addition, we found that monoclonal anti-retinol dehydrogenase antibody bound specifically to retinal pigment epithelium and not to Muller cells or to rod outer segments by immunohistochemical methods.