Frey S E, Hingorani V N, Su-Tsai S M, Ho Y K
Department of Biological Chemistry, University of Illinois, Chicago 60612.
Biochemistry. 1988 Oct 18;27(21):8209-18. doi: 10.1021/bi00421a034.
The exchange-inert Cr(III) beta, gamma-bidentate guanine nucleotide complexes Cr(III)GTP and Cr(III)Gpp(NH)p were used to probe the role of transducin in activating the retinal cGMP cascade. The Cr(III) nucleotide complexes were found to have lower binding affinity for transducin as compared to the Mg2+ complexes. However, the rate of hydrolysis of the transducin-bound Cr(III)GTP was similar to that of Mg(II)GTP. Cr(III)Gpp(NH)p activated the cGMP phosphodiesterase of photolyzed rod outer segment membranes up to 75% of the Mg(II)Gpp(NH)p level but lacked the ability to dissociated the transducin subunits from the rod outer segment membrane. This result implies that the activation of the phosphodiesterase by transducin-GTP complex is a membrane-associated event and the formation of a soluble complex of transducin-GTP with the inhibitory peptide of the phosphodiesterase may not be an obligatory step. Both the delta and lambda screw sense stereoisomers of Cr(III)Gpp(NH)p were capable of activating the cGMP cascade with no apparent stereoselectivity. The nature of the interaction of the metal ion and GTP at the nucleotide-binding site of transducin is discussed together with the results from previous studies using the phosphorothioate GTP analogues [Yamanaka, G., Eckstein, F., & Stryer, L. (1985) Biochemistry 24, 8094-8101] and is compared to the site found in homologous GTP-binding proteins such as elongation factor Tu [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36; la Cour, T.F.M., Nyborg, J., Thirup, S., & Clark, B.F.C. (1985) EMBO J. 4, 2385-2388]. The implications of the observed results on the molecular mechanism of visual signal transduction are discussed.
交换惰性的Cr(III)β,γ-双齿鸟嘌呤核苷酸复合物Cr(III)GTP和Cr(III)Gpp(NH)p被用于探究转导素在激活视网膜cGMP级联反应中的作用。与Mg2+复合物相比，发现Cr(III)核苷酸复合物对转导素的结合亲和力较低。然而，与转导素结合的Cr(III)GTP的水解速率与Mg(II)GTP的水解速率相似。Cr(III)Gpp(NH)p可将光解的视杆外段膜的cGMP磷酸二酯酶激活至Mg(II)Gpp(NH)p水平的75%，但缺乏将转导素亚基从视杆外段膜上解离的能力。这一结果表明，转导素-GTP复合物对磷酸二酯酶的激活是一个与膜相关的事件，转导素-GTP与磷酸二酯酶抑制肽形成可溶性复合物可能不是一个必要步骤。Cr(III)Gpp(NH)p的δ和λ螺旋方向立体异构体均能够激活cGMP级联反应，且没有明显的立体选择性。本文讨论了转导素核苷酸结合位点处金属离子与GTP相互作用的性质，并结合了先前使用硫代磷酸酯GTP类似物的研究结果[Yamanaka, G., Eckstein, F., & Stryer, L. (1985) Biochemistry 24, 8094-8101]，并与在同源GTP结合蛋白如延伸因子Tu中发现的位点进行了比较[Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36; la Cour, T.F.M., Nyborg, J., Thirup, S., & Clark, B.F.C. (1985) EMBO J. 4, 2385-2388]。本文还讨论了观察结果对视觉信号转导分子机制的影响。
