Gollapalli Deviprasad R, Maiti Pranab, Rando Robert R
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115, USA.
Biochemistry. 2003 Oct 14;42(40):11824-30. doi: 10.1021/bi035227w.
RPE65 is a major protein of unknown function found associated with the retinyl pigment epithelial (RPE) membranes [Hamel, C. P., Tsilou, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Biol. Chem. 268, 15751-15757; Bavik, C. O., Levy, F., Hellman, U., Wernstedt, C., and Eriksson, U. (1993) J. Biol. Chem. 268, 20540-20546]. RPE65 knockouts fail to synthesize 11-cis-retinal, the chromophore of rhodopsin, and accumulate all-trans-retinyl esters in the RPE. Previous studies have also shown that RPE65 is specifically labeled with all-trans-retinyl ester based affinity labeling agents, suggesting a retinyl ester binding role for the protein. In the present work, we show that purified RPE65 binds all-trans-retinyl palmitate (tRP) with a K(D) = 20 pM. These quantitative experiments are performed by measuring the quenching of RPE65 fluorescence by added tRP. The binding for tRP is highly specific because 11-cis-retinyl palmitate binds with a K(D) = 14 nM, 11-cis-retinol binds with a K(D) = 3.8 nM, and all-trans-retinol (vitamin A) binds with a K(D) = 10.8 nM. This stereospecificity for tRP is to be compared to the binding of retinoids to BSA, where virtually no discrimination is found in the binding of the same retinoids. This work provides further evidence that RPE65 functions by binding to and mobilizing the highly hydrophobic all-trans-retinyl esters, allowing them to enter the visual cycle.
RPE65是一种功能未知的主要蛋白质，发现它与视网膜色素上皮（RPE）膜相关联[哈梅尔，C.P.，齐洛，E.，普费弗，B.A.，胡克斯，J.J.，德特里克，B.，和雷德蒙德，T.M.（1993年）《生物化学杂志》268卷，第15751 - 15757页；巴维克，C.O.，利维，F.，赫尔曼，U.，韦恩斯特德，C.，和埃里克森，U.（1993年）《生物化学杂志》268卷，第20540 - 20546页]。RPE65基因敲除小鼠无法合成视紫红质的发色团11 - 顺式视黄醛，并在RPE中积累全反式视黄酯。先前的研究还表明，RPE65被基于全反式视黄酯的亲和标记剂特异性标记，这表明该蛋白质具有视黄酯结合作用。在本研究中，我们表明纯化的RPE65以K(D)=20 pM的亲和力结合全反式棕榈酸视黄酯（tRP）。这些定量实验是通过测量添加的tRP对RPE65荧光的淬灭来进行的。tRP的结合具有高度特异性，因为11 - 顺式棕榈酸视黄酯的结合亲和力K(D)=14 nM，11 - 顺式视黄醇的结合亲和力K(D)=3.8 nM，全反式视黄醇（维生素A）的结合亲和力K(D)=10.8 nM。这种对tRP的立体特异性与类视黄醇与牛血清白蛋白（BSA）的结合情况形成对比，在BSA中，相同类视黄醇的结合几乎没有差异。这项工作进一步证明了RPE65通过结合并转运高度疏水的全反式视黄酯来发挥作用，使它们能够进入视觉循环。
