Shukolyukov S A
Sechenov Institute for Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, 194223, Russia.
Biochemistry (Mosc). 2009 Jun;74(6):599-604. doi: 10.1134/s0006297909060029.
After solubilization of frog rod outer segments (ROS) with mild detergents (digitonin, n-dodecyl-beta-D-maltoside, Chaps, Triton X-100) and subsequent one-dimensional blue native polyacrylamide gel electrophoresis (1D BN-PAGE), the position of rhodopsin (Rh) on the gradient gel does not match the monomer with molecular weight of 40 kDa but appears self-associated into aggregate of Rh (RhA) with molecular mass varying in different detergents from 85 to 125 kDa. Short-term treatment (~2 h) of the excised BN-PAGE strip containing RhA by denaturing detergent mixture (10% SDS + 1 mM dithiothreitol (DTT)) followed by 2D SDS-PAGE revealed dissociation of the RhA into opsin monomer and unidentified proteins. Long-term treatment (approximately 2 days) of RhA that included extraction, denaturation, concentration, and electrophoresis induced, along with dissociation of RhA into opsin monomer + unidentified proteins, also formation of opsin dimers, trimers, and higher oligomers owing to a secondary aggregation of opsin. Direct solubilization of the ROS by harsh SDS + DTT detergent mixture followed by 1D SDS-PAGE revealed only opsin monomer that upon heating disappeared, transforming into higher oligomers owing to secondary aggregation. The data show that degree of Rh oligomerization depends on specific conditions in which it stays. In the native state in the photoreceptor membrane as well as in mild detergents frog Rh exists mainly as dimers or higher oligomers. After solubilization with denaturing detergents, RhA can dissociate into monomers that then spontaneously self-associate into higher oligomers under the influence of various factors (for example, heating).
用温和去污剂（洋地黄皂苷、正十二烷基-β-D-麦芽糖苷、CHAPS、 Triton X-100）溶解青蛙视杆细胞外段（ROS），随后进行一维蓝色非变性聚丙烯酰胺凝胶电泳（1D BN-PAGE），视紫红质（Rh）在梯度凝胶上的位置与分子量为40 kDa的单体不匹配，而是呈现自我缔合形成视紫红质聚集体（RhA），其分子量在不同去污剂中从85 kDa到125 kDa不等。用变性去污剂混合物（10% SDS + 1 mM二硫苏糖醇（DTT））对含有RhA的切除的BN-PAGE条带进行短期处理（约2小时），然后进行二维SDS-PAGE，结果显示RhA解离为视蛋白单体和未鉴定的蛋白质。对RhA进行长期处理（约2天），包括提取、变性、浓缩和电泳，除了RhA解离为视蛋白单体 + 未鉴定的蛋白质外，还由于视蛋白的二次聚集形成了视蛋白二聚体、三聚体和更高的寡聚体。用苛刻的SDS + DTT去污剂混合物直接溶解ROS，随后进行一维SDS-PAGE，结果显示只有视蛋白单体，加热后消失，由于二次聚集转化为更高的寡聚体。数据表明，Rh寡聚化程度取决于其所处的特定条件。在光感受器膜的天然状态以及温和去污剂中，青蛙Rh主要以二聚体或更高的寡聚体形式存在。用变性去污剂溶解后，RhA可以解离为单体，然后在各种因素（例如加热）的影响下自发地自我缔合形成更高的寡聚体。
