Activation mechanism of retinal rod cyclic GMP phosphodiesterase probed by fluorescein-labeled inhibitory subunit.
Activation mechanism of retinal rod cyclic GMP phosphodiesterase probed by fluorescein-labeled inhibitory subunit.
复制标题
荧光素标记抑制亚基探测视网膜杆环 GMP 磷酸二酯酶的激活机制。
DOI：
10.1021/bi00460a028
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Stryer,L
中科院分区：
文献类型：
--
作者：
Wensel,TG;Stryer,L
斯坦福大学医学院细胞生物学系，Sherman Fairchild Center，Stanford，California 94305 收稿日期：1989 年 7 月 20 日；修订稿于 1989 年 10 月 13 日收到摘要：脊椎动物视网膜 Rodouter 节段 (ROS) 的环 GMP 磷酸二酯酶 (PDE) 在黑暗中通过其 7 个亚基保持不活跃，并在转导蛋白 (Ta-GTP) 亚基的 GTP 形式照射后被激活。最近的研究表明，受抑制的全酶的化学计量为a/？ 72. Ta-GTP 和 7 相互作用。我们使用荧光素标记的7亚基（7F）作为探针研究了激活机制。通过 PDE 与 5-(碘乙酰胺)荧光素反应并通过反相高压液相色谱 (HPLC) 纯化，制备含有单一共价连接荧光素的 7F。 7F 与天然 7 一样，抑制胰蛋白酶激活的 PDE 和转导蛋白激活的 PDE 的催化活性。通过进一步添加 Ta-GTP 克服了 7F 的抑制作用。 7F 与剥离了 PDE 和其他外周膜蛋白的 ROS 膜的结合非常弱。添加到 ROS 膜中的 7F 并入可以用低离子强度缓冲液提取的成分中。 HPLC凝胶过滤显示7F成为PDE全酶的一部分。在有光和 GTP 存在的情况下，掺入发生在不到 1 分钟内，但在没有 GTP 的情况下，掺入速度要慢得多（Z1/2~ 500 s）。该结果表明转导蛋白通过与全酶结合并加速 7 从抑制位点解离来激活 PDE。通过稳态发射各向异性测量来监测 7F 与胰蛋白酶激活的 PDEa# 3 的结合，并与 PDE 活性进行比较。结果表明，每 7 个结合位点的占据抑制了 PDEa 总活性的大约一半 (J；这些位点的解离常数相似 (~ 10 pM)。我们的结果符合一个简单的模型，其中 Ta-GTP 首先与 afiyy 全酶相互作用并带走 7 个亚基之一，形成部分活性^复合物。然后，该复合物可以转化为完全活性的 PDEa（当第二个 7 亚基存在时为 S） 被另一个Ta-GTP带走了。 TQ-PDE7 复合物保持与膜结合。
Department of Cell Biology, Stanford University School of Medicine, Sherman Fairchild Center, Stanford, California 94305 Received July 20, 1989; Revised Manuscript Received October 13, 1989 abstract: The cyclic GMP phosphodiesterase (PDE) of vertebrate retinal rodouter segments (ROS) is kept inactive in the dark by its 7 subunits and is activated followingillumination by the GTP form of the a subunit of transducin (Ta-GTP). Recentstudies have shown that the stoichiometry of the inhibited holoenzyme is a/? 72. Ta-GTP and 7 act reciprocally. We have investigated the activation mechanism using fluorescein-labeled 7 subunit (7F) as a probe. 7F containing a single covalently attached fluorescein was prepared by reaction of PDE with 5-(iodoacetamido) fluorescein and purification by reversed-phase high-pressure liquid chromatography (HPLC). 7F, like native 7, inhibits the catalytic activity of trypsin-activated PDE and transducin-activated PDE. Inhibition by 7F was overcome by further addition of Ta-GTP. 7F binds very weakly to ROS membranes stripped of PDE and other peripheral membrane proteins. 7F added to ROS membranes became incorporated into a component that could be extracted with a low ionic strength buffer. HPLC gel filtration showed that 7F became part of the PDE holoenzyme. Incorporation occurred in less than 1 min in the presence of light and GTP, but much more slowly (Z1/2~ 500 s) in the absence of GTP. This result indicates that transducin activates PDE by binding to the holoenzyme and accelerating the dissociation of 7 from the inhibitory sites. The binding of 7F to trypsin-activated PDEa# 3 was monitored by steady-state emissionanisotropy measurements and compared with PDE activity. The results indicate that occupancy of each 7 binding site suppresses about half of the total activity of PDEa (J; the dissociation constants for these sites are similar (~ 10 pM). Our results fit a simple model in which Ta-GTP interacts first with an afiyy holoenzyme and carries away one of the 7 subunits, to form a partially active^ complex. This complex can then be converted to fully active PDEa (S when a second 7 subunit is carried away by anotherTa-GTP. The TQ-PDE7 complex stays bound to the membrane.