Phillips W J, Cerione R A
Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853-6401.
J Biol Chem. 1988 Oct 25;263(30):15498-505.
We have made use of the enhancement of the intrinsic fluorescence of the alpha subunit of transducin (alpha T), which accompanies guanine nucleotide exchange, to follow the reconstituted interactions between pure rhodopsin and pure transducin in phospholipid vesicles. When the pure alpha T.GDP complex is added to lipid vesicles containing rhodopsin and the beta gamma T complex, a light- and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-dependent enhancement of the fluorescence emission of alpha T is observed. When GTP is substituted for GTP gamma S, a similar enhancement of the intrinsic fluorescence of alpha T occurs; however, this enhancement is transient and precedes a fluorescence decay which is complete in 2-5 min. The fact that the fluorescence decay is specifically induced by GTP and is not observed either with nonhydrolyzable GTP analogs or with NaF (plus AlCl3) indicates that the decay represents GTP hydrolysis in alpha T. The dose-response profiles for the effects of the beta gamma T complex on the rate and extent of the GTP gamma S-stimulated fluorescence enhancement of alpha T have also been examined. The addition of relatively low levels of beta gamma T to these reconstituted systems can promote the GTP gamma S-stimulated enhancement of the fluorescence of multiple alpha T subunits with half-maximal enhancement occurring at alpha T:beta gamma T ratios of 150:1. These findings are consistent with earlier suggestions (Fung, B. K.-K. (1983) J. Biol. Chem. 258, 10495-10502) that the beta gamma T subunit dissociates from alpha T as a result of the GDP-GTP exchange reaction and thus can act catalytically to promote the activation of a number of inactive alpha T species. However, the dependence of the rate of the GTP gamma S-stimulated fluorescence enhancement on beta gamma T is complex and cannot be explained adequately by simple models where alpha T-beta gamma T interactions (or rhodopsin-transducin interactions) are rate-limiting for the rhodopsin-stimulated activation of the alpha T subunits. Overall, the results reported here demonstrate that fluorescence spectroscopy can be used to monitor directly a receptor-catalyzed activation-deactivation cycle of a GTP-binding protein within a lipid milieu.
我们利用转导素α亚基（αT）内在荧光的增强（伴随鸟嘌呤核苷酸交换），来追踪磷脂囊泡中纯视紫红质与纯转导素之间重构的相互作用。当将纯αT·GDP复合物添加到含有视紫红质和βγT复合物的脂质囊泡中时，可观察到αT荧光发射的光和鸟苷5'-O-(3-硫代三磷酸)(GTPγS)依赖性增强。当用GTP替代GTPγS时，αT的内在荧光也会出现类似的增强；然而，这种增强是短暂的，且在2-5分钟内荧光完全衰减之前出现。荧光衰减是由GTP特异性诱导的，在不可水解的GTP类似物或NaF（加AlCl3）存在下均未观察到，这一事实表明衰减代表αT中的GTP水解。还研究了βγT复合物对GTPγS刺激的αT荧光增强速率和程度影响的剂量反应曲线。向这些重构系统中添加相对低水平的βγT可以促进多个αT亚基的GTPγS刺激的荧光增强，在αT:βγT比例为150:1时出现半数最大增强。这些发现与早期的推测一致（冯，B.K.-K.（1983年）《生物化学杂志》258，10495-10502），即βγT亚基由于GDP-GTP交换反应而与αT解离，因此可以催化促进许多无活性αT物种的激活。然而，GTPγS刺激的荧光增强速率对βγT的依赖性很复杂，简单的模型（其中αT-βγT相互作用（或视紫红质-转导素相互作用）对视紫红质刺激的αT亚基激活起限速作用）无法充分解释。总体而言，此处报道的结果表明荧光光谱可用于直接监测脂质环境中GTP结合蛋白的受体催化激活-失活循环。
