Gray-Keller M P, Biernbaum M S, Bownds M D
Neuroscience Training Program, University of Wisconsin, Madison 53706.
J Biol Chem. 1990 Sep 5;265(25):15323-32.
An electropermeabilized preparation of frog retinal rod outer segments (ROS) has been developed to examine the light sensitivity and amplification of visual transduction reactions in a minimally disturbed environment. Electropermeabilized ROS are indistinguishable from whole and osmotically intact ROS in the light microscope and retain 3-fold more protein than mechanically disrupted ROS. They differ from mechanically fragmented ROS in several respects. Illumination results in more amplified activation of the GTP-binding protein transducin (Gt) than previously observed: bleaching as little as approximately 1 rhodopsin molecule (Rho*) in every 10 disks within a single ROS activates 37,000 molecules of Gt per Rho*, equivalent to 70% of the light-activatable Gt present on a single disk face. This amplification is maintained over approximately 1 decade of light intensity but drops sharply as disk faces begin to absorb a second photon. Lower amplification is observed in fragmented ROS and derives from the fact that physical disruption of ROS causes Gt to bind GTP and elute from the membrane, thus decreasing the amount remaining and available for light activation. Illumination of electropermeabilized ROS in the presence of GTP or of the nonhydrolyzable substrate guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) causes redistribution of Gt: an amount (approximately 20 mmol/mol Rho) equivalent to the amount of inhibitory gamma subunit of phosphodiesterase (PDE) remains internal and bound to nucleotide, and the remaining activated Gt diffuses out in a manner graded with light intensity. This suggests that PDE activation by Gt alpha may not require dissociation of Gt alpha bound to the gamma subunit of PDE in a form than can elute from ROS. Two further differences between electropermeabilized and mechanically disrupted ROS are noted: the addition of ATP to electropermeabilized ROS does not affect the light sensitivity or kinetics of the GTP binding reaction, and a specificity for light-induced GTP versus GDP binding is observed.
已开发出一种经电通透处理的青蛙视网膜视杆细胞外段（ROS）制剂，用于在最小程度受干扰的环境中检测视觉转导反应的光敏感性和放大作用。在光学显微镜下，经电通透处理的ROS与完整且渗透压完整的ROS无法区分，并且比机械破碎的ROS保留的蛋白质多3倍。它们在几个方面与机械破碎的ROS不同。光照导致GTP结合蛋白转导素（Gt）的激活比以前观察到的更具放大作用：在单个ROS内每10个盘片中漂白低至约1个视紫红质分子（Rho*），每个Rho*可激活37,000个Gt分子，相当于单个盘面存在的可光激活Gt的70%。这种放大作用在大约1个光强 decade 范围内保持，但随着盘面开始吸收第二个光子而急剧下降。在破碎的ROS中观察到较低的放大作用，这是因为ROS的物理破坏导致Gt结合GTP并从膜上洗脱，从而减少了剩余并可用于光激活的量。在GTP或不可水解底物鸟苷5'-（γ-硫代）三磷酸（GTPγS）存在下照射经电通透处理的ROS会导致Gt重新分布：相当于磷酸二酯酶（PDE）抑制性γ亚基量（约20 mmol/mol Rho）的量保持在内部并与核苷酸结合，其余活化的Gt以与光强分级的方式扩散出去。这表明Gtα对PDE的激活可能不需要以可从ROS洗脱的形式与PDE的γ亚基结合的Gtα解离。还注意到经电通透处理的ROS和机械破碎的ROS之间的另外两个差异：向经电通透处理的ROS中添加ATP不会影响GTP结合反应的光敏感性或动力学，并且观察到光诱导的GTP与GDP结合的特异性。
