INTERACTION BETWEEN THE RETINAL CYCLIC-GMP PHOSPHODIESTERASE INHIBITOR AND TRANSDUCIN - KINETICS AND AFFINITY STUDIES
INTERACTION BETWEEN THE RETINAL CYCLIC-GMP PHOSPHODIESTERASE INHIBITOR AND TRANSDUCIN - KINETICS AND AFFINITY STUDIES
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DOI：
10.1021/bi00084a035
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发表时间：
1993-08-24
期刊：
影响因子：
2.9
通讯作者：
CHABRE, M
中科院分区：
文献类型：
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作者：
OTTOBRUC, A;ANTONNY, B;CHABRE, M
在视网膜环GMP磷酸二酯酶（PDE）中，α -异二聚体的催化作用在黑暗中被两个相同的γ -亚基抑制，而在光照下被异三聚体g蛋白转导蛋白（tbetagama - talphagdp）的携带gtp的α -亚基刺激。两个TalphaGTP分子，从PDEalphabeta解离，结合并取代PDEalphabeta上的PDEgamma亚基抑制位点。用GTPgammaS代替GTP，这种联系变得持久。在生理条件下，PDEalphabeta(γ - α)2活性复合物停留在细胞膜上。但在低盐缓冲液中，它变得可溶并解离成部分活性的PDEalphabeta催化片段和两个pdegamma - talphagtpgamma复合物。这表明Talpha优先与PDEgamma结合。我们研究了重组牛PDEgamma与纯化的Talpha溶液或与含有Tbetagamma-TalphaGDP和PDEalphabetagamma2的视网膜杆外段（ROS）的相互作用。当添加到暗ROS中时，重组PDEgamma不与无活性的PDEalphabetagamma2结合，而是从膜结合的全转导蛋白中提取TalphaGDP，形成可溶的PDEgamma-TalphaGDP复合物。在溶液中，PDEgamma也与纯化的TalphaGDP结合。通过监测蛋白质色氨酸荧光的变化来确定PDEgamma与TalphaGDP或TalphaGTPgammaS相互作用的动力学和亲和性。重组PDEgamma与可溶性TalphaGTPgammaS和TalphaGDP结合的K(d) s分别小于等于0.1 nM和3 nM。PDEgamma-TalphaGDP在3秒内解体。这种缓慢的解离意味着，在原位，talpa -PDEgamma不能物理地离开活性的PDEalphabeta，因为在GTP水解后，分离的talpa -PDEgamma复合物解离太慢，无法允许释放的PDEgamma快速抑制PDE。将重组PDEgamma加入到被TalphaGTPgammaS持续激活的PDE中，抑制发生，TalphaGTPgammaS与天然PDEgamma复合物释放，表明两者迄今为止一直与PDEalphabeta结合。突变W70F不会阻止重组PDEgamma抑制PDEalphabeta，但会使其对TalphaGTP和TalphaGDP的亲和力降低100倍。因此，PDEgamma的W70必须参与其与TalphaGTP和TalphaGDP的结合。当用[W70F]PDEgamma重组后，PDE被完全抑制，而不再被TalphaGTP或TalphaGTP- gammas激活。
In the retinal cyclic GMP phosphodiesterase (PDE), catalysis by the alphabeta-heterodimer is inhibited in the dark by two identical gamma-subunits and stimulated in the light by the GTP-bearing alpha-subunit of the heterotrimeric G-protein transducin (Tbetagamma-TalphaGDP). Two TalphaGTP molecules, dissociated from Tbetagamma,bind to and displace the PDEgamma subunits from their inhibitory sites on PDEalphabeta. With GTPgammaS in lieu of GTP, this association becomes persistent. Under physiological conditions, the PDEalphabeta(gammaTalpha)2 active complex stays on the membrane. But in low-salt buffers, it becomes soluble and dissociates into a partially active PDEalphabeta catalytic moiety and two PDEgamma-TalphaGTPgammaS complexes. This indicates that Talpha binds preferentially to PDEgamma. We have studied the interaction of recombinant bovine PDEgamma with purified Talpha in solution or with retinal rod outer segments (ROS) containing both Tbetagamma-TalphaGDP and PDEalphabetagamma2. When added to dark ROS, recombinant PDEgamma did not bind to inactive PDEalphabetagamma2 but extracted TalphaGDP from membrane-bound holo-transducin to form a soluble PDEgamma-TalphaGDP complex. PDEgamma also bound to purified TalphaGDP in solution. The kinetics and affinity of the interaction between PDEgamma and TalphaGDP or TalphaGTPgammaS were determined by monitoring changes in the proteins' tryptophan fluorescence. The K(d)'s for the binding of recombinant PDEgamma to soluble TalphaGTPgammaS and TalphaGDP are less-than-or-equal-to 0.1 and 3 nM, respectively. PDEgamma-TalphaGDP falls apart in 3 s. This slow dissociation means that, in situ, Talpha-PDEgamma cannot physically leave the active PDEalphabeta, since after GTP hydrolysis, an isolated Talpha-PDEgamma complex would dissociate too slowly to allow a fast PDE reinhibition by the liberated PDEgamma. When recombinant PDEgamma was added to PDE that had been persistently activated by TalphaGTPgammaS, reinhibition occurred and TalphaGTPgammaS, complexed to the native PDEgamma, was released, indicating that both had hitherto stayed bound to PDEalphabeta. The mutation W70F does not prevent recombinant PDEgamma from inhibiting PDEalphabeta but diminishes its affinity for TalphaGTP and TalphaGDP 100-fold. Thus, W70 of PDEgamma must take part in its binding to TalphaGTP and TalphaGDP. When reconstituted with [W70F]PDEgamma, PDE was fully inhibited but could no longer be activated by TalphaGTP or TalphaGTP-gammaS.