牛视网膜色素上皮中视黄醇脱氢酶的鉴定及免疫组织化学研究

Identification and immunohistochemistry of retinol dehydrogenase from bovine retinal pigment epithelium.

作者信息

Suzuki Y, Ishiguro S, Tamai M

机构信息

Department of Ophthalmology, Tohoku University School of Medicine, Sendai, Japan.

出版信息

Biochim Biophys Acta. 1993 May 13;1163(2):201-8. doi: 10.1016/0167-4838(93)90182-q.

DOI:10.1016/0167-4838(93)90182-q

PMID:8490052

Abstract

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 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 Müller cells or to rod outer segments by immunohistochemical methods.

摘要

我们研究了来自牛视网膜色素上皮的视黄醇脱氢酶（11-顺式特异性）的特性。去污剂会导致视黄醇脱氢酶活性丧失；因此，我们添加3 mM NADH作为稳定剂来溶解这种酶，并使用琼脂糖CL-6B和羟基磷灰石柱色谱对该酶进行部分纯化。部分纯化的样品含有两种主要蛋白质（66 kDa，33 kDa），对11-顺式和13-顺式视黄醛有底物偏好，而对全反式和9-顺式异构体没有偏好。通过蛋白质印迹法检测视网膜色素上皮粗提物的单克隆抗33 kDa蛋白。此外，我们通过免疫组织化学方法发现，单克隆抗视黄醇脱氢酶抗体特异性结合视网膜色素上皮，而不结合米勒细胞或视杆外段。