Efficiencies of Activation of Transducin by Cone and Rod Visual Pigments
Efficiencies of Activation of Transducin by Cone and Rod Visual Pigments
复制标题
DOI：
10.1021/bi3015967
复制
发表时间：
2013-04-30
期刊：
影响因子：
2.9
通讯作者：
Shichida, Yoshinori
中科院分区：
文献类型：
--
作者：
Imamoto, Yasushi;Seki, Ichirota;Shichida, Yoshinori
由于 Gt 激活状态 (Meta-II)，光诱导转导蛋白 (Gt) 激活过程在视锥视色素和视杆视色素（视紫红质）之间的生化差异仍不清楚。视锥细胞视色素的衰减速度太快，无法通过传统的生化方法（例如 GTP gamma S 结合测定）精确测量激活效率。在这里，我们通过监测色素和Gt中色氨酸残基的固有荧光来实时测量鸡绿色敏感视锥视色素（cG）和牛视紫红质（bRh）的激活效率。 Gt 激活的 Michaelis-Menten 分析表明，cG 的初始速度大约是 bRh 的一半，而它们的米氏常数相当。由于Meta-II中视网膜席夫碱的快速水解，cG对Gt的激活立即减慢，但这种水解通过与Gt形成复合物而被抑制。使用位置 122 和 189 的 cG 和 bRh 突变体（其在 Meta-II 中表现出发色团水解速率的改变），我们发现 Gt 激活的初始速度与发色团水解速率呈负相关。这些结果表明，122 和 189 位的氨基酸残基不仅解释了 Meta-II 中对发色团水解的抗性，而且还解释了 Meta-II 有效 Gt 激活的构象。当棒响应单个光子时，Rh 的 Gt 激活态的寿命要长得多，才能抑制由 Gt 激活态的随机衰变引起的自发猝灭。
How the light-induced transducin (Gt) activation process differs biochemically between cone visual pigments and rod visual pigment (rhodopsin) has remained unclear, because the Gt-activating state (Meta-II). of cone visual pigment decays too fast to precisely measure the activation efficiency by conventional biochemical methods such as the GTP gamma S binding assay. Here we measured the activation efficiencies of chicken green-sensitive cone visual pigment (cG) and bovine rhodopsin (bRh) in real time by monitoring the intrinsic fluorescence of tryptophan residues in the pigments and Gt. Michaelis-Menten analysis of Gt activation showed that the initial velocity for cG was approximately half that for bRh, while their Michaelis constants were comparable. Gt activation by cG was immediately slowed because of the fast hydrolysis of the retinal Schiff base in Meta-II, but this hydrolysis was suppressed by forming the complex with Gt. Using mutants of cG and bRh for positions 122 and 189, which exhibit altered rates of chromophore hydrolysis in Meta-II, we found that the initial velocity of Gt activation is negatively correlated with the rate of chromophore hydrolysis. These results suggest that the amino acid residues at positions 122 and 189 account for not only the resistance to the chromophore hydrolysis in Meta-II but also the conformation of Meta-II for efficient Gt activation. The substantially longer lifetime of the Gt activating state of Rh would be necessary to suppress the spontaneous quenching by the stochastic decay of the Gt-activating state when a rod responds to a single photon.