Characterization of Transducin from Bovine Retinal Rod Outer Segments
Characterization of Transducin from Bovine Retinal Rod Outer Segments
复制标题
牛视网膜杆外节转导蛋白的表征
DOI：
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发表时间：
2001
期刊：
影响因子：
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通讯作者：
K.K
中科院分区：
文献类型：
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作者：
E.;K.K
转导蛋白是一种鸟嘌呤核苷酸结合蛋白，由两个亚基（Tu和TB7）组成，介导视紫红质与视网膜杆外节膜结合的环GMP磷酸二酯酶之间的信号耦合。To亚基是磷酸二酯酶的激活剂，TB7亚基的功能是将Tu与磷酸化的视紫红质物理连接起来。在这项研究中，在一个由纯化的转导蛋白和剥离棒外段膜组成的重组系统中，研究了霍乱毒素催化Tu的adp核糖基化机制。胰蛋白酶对标记的Tu进行有限的蛋白水解，表明插入的adp核糖完全位于单个蛋白水解片段上，表观分子量为23,000。当鸟苷5′-（B，y-亚胺）三磷酸（Gpp(NH)p）和TB7的浓度与Tu相同时，紫红质在400-500 nm区域连续照射时，ADPribose的掺入量最大。光照的刺激作用与视网膜发色团与视蛋白的直接相互作用有关。这些发现有力地表明，由T、*Gpp(NH)p、TB7和紫红质光中间体组成的瞬时蛋白复合物是霍乱毒素所需的底物。单次转换动力学测量表明，T的adp核糖基化与GTP水解速率非常慢的转导蛋白分子群的出现一致。该群体结合GTP的水解速率为1.1% 10-3/s，比未修饰的转导蛋白的水解速率慢22倍。
Transducin, a guanine nucleotide-binding protein consisting of two subunits (Tu and TB7), mediates the signal coupling between rhodopsin and a membranebound cyclic GMP phosphodiesterase in retinal rod outer segments. The To subunit is an activator of the phosphodiesterase, and the function of the TB7 subunit is to physically link Tu with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of Tu has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled Tu with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADPribose was achieved when guanosine 5‘-(B,y-imido)triphosphate (Gpp(NH)p) and TB7 were present at concentrations equal to that of Tu and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T,*Gpp(NH)p, TB7, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T, coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 % 10-3/s, which was 22-fold slower than the rate for the unmodified transducin.