Departamento de Biología Celular, División de Ciencias Biológicas, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Baruta, Caracas 1081-A, Venezuela.
Arch Biochem Biophys. 2011 Mar 15;507(2):219-31. doi: 10.1016/j.abb.2010.12.018. Epub 2010 Dec 19.
A 50-kDa-polypeptide band peripherally bound to retinal rod outer segment (ROS) membranes was purified by anion-exchange chromatography. When the 50-kDa protein was compared with purified arrestin-1, it was observed that: (1) both proteins comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and were recognized by either anti-50-kDa protein polyclonal antibodies or anti-arrestin-1 monoclonal antibodies; (2) protein fragments and peptide fingerprint maps obtained following limited and complete proteolysis with specific proteases were very similar for both molecules; and (3) several chromatographically-purified tryptic peptides from the 50-kDa protein possessed the same amino acid composition as tryptic peptides deduced from the reported arrestin-1 primary structure. Consequently, arrestin-1 and the purified 50-kDa protein must correspond to variants of the same molecule. However, in contrast to arrestin-1 that associated to the ROS membranes only in the presence of light and ATP, the 50-kDa protein interacted with the ROS membranes in a light-independent manner, either in the presence or absence of ATP. These results clearly established that phosphorylated and illuminated rhodopsin is not the membrane anchor for this variant of arrestin-1.
一种 50kDa 多肤带外周结合到视网膜杆状细胞外节（ROS）膜上，通过阴离子交换层析被纯化。当该 50kDa 蛋白与纯化的 arrestin-1 比较时，发现：（1）两种蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上共迁移，并且都被抗 50kDa 蛋白多克隆抗体或抗 arrestin-1 单克隆抗体识别；（2）用特定蛋白酶进行有限和完全蛋白水解后获得的蛋白片段和肽指纹图谱对于两种分子非常相似；（3）从 50kDa 蛋白中分离出的几种经色谱纯化的胰蛋白酶肽与从报道的 arrestin-1 一级结构推导的胰蛋白酶肽具有相同的氨基酸组成。因此，arrestin-1 和纯化的 50kDa 蛋白必须对应于同一分子的变异体。然而，与仅在光照和 ATP 存在下与 ROS 膜结合的 arrestin-1 相反，50kDa 蛋白以非光照依赖的方式与 ROS 膜相互作用，无论是否存在 ATP。这些结果清楚地表明，磷酸化和光照视紫红质不是该 arrestin-1 变体的膜锚定物。
