A cleavable affinity biotinylating agent reveals a retinoid binding role for RPE65
A cleavable affinity biotinylating agent reveals a retinoid binding role for RPE65
复制标题
DOI：
10.1021/bi034002i
复制
发表时间：
2003-05-27
期刊：
影响因子：
2.9
通讯作者：
Rando, RR
中科院分区：
文献类型：
--
作者：
Jahng, WJ;David, C;Rando, RR
视网膜色素上皮细胞（RPE）膜含有完整的生化装置，能够将全反式视黄醇（维生素A）加工成11-顺式-视网膜，即视觉发色团。由于这些蛋白质中的许多是整合的膜蛋白质并且对传统鉴定方法具有抗性，因此寻找鉴定这些蛋白质的替代方法。本文所述的方法涉及用碱可切割接头的亲和生物素化。描述了一种含有维生素A的亲和标记卤代乙酸盐，其有助于类维生素A结合蛋白（RBP）的鉴定。用低微摩尔浓度的（3R）-3-[boc-lys（biotinyl）-O]-all-trans-retinol chloroacetate I处理粗制牛RPE膜导致RPE 65和卵磷脂视黄醇酰基转移酶（LRAT）的特异性标记。仅RPE 65在4 ℃以5 μ M-1标记。通过将标记的蛋白质结合到含有抗生物素蛋白的珠上，随后在pH 11下从珠上切割蛋白质，容易地分离标记的RPE 65。胰蛋白酶消化经1.随后进行质谱分析，证明C231和C448被1烷基化。这些研究验证了所使用的方法，并且进一步证明RPE 65（RPE的主要膜相关蛋白）是RBP。
Retinal pigment epithelial (RPE) membranes contain the full biochemical apparatus capable of processing all-trans-retinol (vitamin A) into 11-cis-retinal, the visual chromophore. As many of these proteins are integral membrane proteins and resistant to traditional methods of identification, alternate methods of identifying these proteins are sought. The approach described here involves affinity biotinylation with alkali cleavable linkers. A vitamin A containing affinity-labeling haloacetate is described which facilitates the identification of retinoid binding proteins (RBPs). Treatment of crude bovine RPE membranes with (3R)-3-[boc-lys(biotinyl)-O]-all-trans-retinol chloroacetate I in the low micromolar range led to the specific labeling of RPE65 and lecithin retinol acyltransferase (LRAT). Only RPE65 is labeled at 5 muM 1 at 4 degreesC. Labeled RPE65 was readily isolated by binding the labeled protein to avidin-containing beads, followed by cleavage of the protein from the beads at pH 11. Trypsin digestion of RPE65 modified by 1. followed by mass spectrometry, demonstrates that C231 and C448 are alkylated by 1. These studies validate the approach that was used, and furthermore demonstrate that RPE65, a major membrane-associated protein of the RPE, is a RBP.