Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina.
Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina.
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DOI：
10.1083/jcb.97.3.703
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发表时间：
1983-09
期刊：
影响因子：
--
通讯作者：
Saari JC
中科院分区：
文献类型：
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作者：
Bunt-Milam AH;Saari JC
最近发现和表征了几种用内源性结合类维生素A纯化的蛋白质，这表明这些在视网膜中丰富的蛋白质在维生素A的运输和功能中起作用。免疫细胞化学技术被用来定位两个维甲酸结合蛋白在视网膜的四个物种。用牛视网膜纯化抗原免疫家兔，获得细胞视网膜结合蛋白（CRALBP）和感光细胞间类视黄酸结合蛋白（IRBP）的抗血清。来自每种抗血清的抗体与视网膜匀浆和上清液中的单一组分反应，其对应于相应抗原的分子量和电荷（非SDS和SDS PAGE，电泳转移至硝酸纤维素，免疫化学染色）。免疫细胞化学对照是来自非免疫血清的抗体和用纯化抗原吸收的抗体。使用免疫荧光和过氧化物酶-抗过氧化物酶技术，将抗原定位于牛、大鼠、猴和人视网膜的冷冻切片上。在感光细胞外节周围的空间中发现了抗IRBP的特异性染色，其中最重的标记在对应于视网膜色素上皮（RPE）顶面的线中。锥体外段阳性。在所有物种的两种细胞类型中发现了抗CRALBP染色：RPE和Muller神经胶质细胞。在RPE内，标记充满细胞质，顶部最重，核阴性。Muller细胞的标记产生了高尔基体样轮廓，所有细胞质隔室都被强烈染色。外界膜染色较重，标记的微绒毛伸入感光细胞间隙。IRBP定位于三种细胞类型（RPE，感光细胞和Muller）的空间，这与其在细胞间类维生素A转运中的作用一致。CRALBP在RPE中的定位证实了以前的生化研究;其在穆勒细胞中的存在表明，这种神经胶质细胞可能在视网膜中的维生素A代谢中发挥迄今为止未被怀疑的作用。
The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Muller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Muller cells produced Golgi- like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Muller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Muller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.