Taiji M, Yokoyama S, Miyazawa T
J Biochem. 1985 Dec;98(6):1447-53. doi: 10.1093/oxfordjournals.jbchem.a135413.
2' and 3'-O-(N-acetyl-L-phenylalanyl)adenosine (Ac-Phe-Ado) were chemically synthesized. These two isomers were clearly separated from each other by high-performance liquid chromatography (HPLC). From the two isomers of [3H]Phe-tRNA in equilibrium, Ac-[3H]Phe-Ado was prepared, without any change in the 2'/3'-isomer ratio, by acetylation of the phenylalanyl residue with acetic anhydride followed by digestion with pancreatic RNase A. By HPLC analysis of this preparation of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was found to be 0.20:0.80. Further, [3H]Phe-tRNA was bound to Escherichia coli polypeptide chain elongation factor Tu (EF-Tu) with the ligand of GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GMP-P(NH)P). The ternary complex was treated with phenol and acetic anhydride, and then digested with pancreatic RNase A. By HPLC analysis of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was determined to be 0.07:0.93 in the complex with EF-Tu.GTP and 0.04:0.96 in the complex with EF-Tu.GMP-P(NH)P. These results clearly indicate that the 3'-isomer, rather than the 2'-isomer, of aminoacyl-tRNA is exclusively involved in the ternary complex.
化学合成了2'-O-(N-乙酰-L-苯丙氨酰)腺苷和3'-O-(N-乙酰-L-苯丙氨酰)腺苷（Ac-Phe-Ado）。通过高效液相色谱（HPLC）可清晰地将这两种异构体彼此分离。从处于平衡状态的[3H]苯丙氨酰-tRNA的两种异构体出发，通过用乙酸酐对苯丙氨酰残基进行乙酰化，随后用胰核糖核酸酶A消化，制备得到了Ac-[3H]Phe-Ado，且2'/3'-异构体比例未发生任何变化。通过对该Ac-[3H]Phe-Ado制剂进行HPLC分析，发现[3H]苯丙氨酰-tRNA的2'-异构体与3'-异构体的丰度比为0.20:0.80。此外，[3H]苯丙氨酰-tRNA与大肠杆菌多肽链延伸因子Tu（EF-Tu）结合，配体为GTP或鸟苷5'-[β,γ-亚氨基]三磷酸（GMP-P(NH)P）。将三元复合物用苯酚和乙酸酐处理，然后用胰核糖核酸酶A消化。通过对Ac-[3H]Phe-Ado进行HPLC分析，确定在与EF-Tu·GTP形成的复合物中，[3H]苯丙氨酰-tRNA的2'-异构体与3'-异构体的丰度比为0.07:0.93，在与EF-Tu·GMP-P(NH)P形成的复合物中为0.04:0.96。这些结果清楚地表明，氨酰-tRNA的3'-异构体而非2'-异构体专门参与三元复合物的形成。
