Tanada S, Kawakami M, Yoneda T, Takemura S
J Biochem. 1981 May;89(5):1565-72. doi: 10.1093/oxfordjournals.jbchem.a133350.
Jekowsky et al. reported recently that elongation factor Tu:GTP complex from Escherichia coli protected aminoacyl-tRNA from digestion by pancreatic RNase (I). On the basis of their finding, we have developed the "RNase-resistance assay" for determination of the dissociation constant of aminoacyl-tRNA from aminoacyl-tRNA:EF-Tu:GTP complex. By the use of this sensitive assay, the dissociation constants were estimated to be 3.6 x 10(-7) M for Ala-tRNA1Ala (Torulopsis utilis), 7.9 x 10(-8) M for Phe-tRNAPhe (Escherichia coli), 8.1 x 10(-7) M for initiator Met-tRNAfMet (Escherichia coli), and 5.4 x 10(-6) M for Gly-tRNA1Gly (Staphylococcus epidermidis) participating in cell wall biosynthesis. Moreover, using a relatively large amount of EF-Tu:GTP, we have been able to detect the ternary complexes of initiator Met-tRNAfMet and Gly-tRNA1Gly with EF-Tu:GTP even by the method of gel filtration.
杰科夫斯基等人最近报道，来自大肠杆菌的延伸因子Tu:GTP复合物可保护氨酰-tRNA不被胰核糖核酸酶（I）消化。基于他们的这一发现，我们开发了“核糖核酸酶抗性测定法”，用于测定氨酰-tRNA从氨酰-tRNA:EF-Tu:GTP复合物中的解离常数。通过使用这种灵敏的测定法，参与细胞壁生物合成的丙氨酰-tRNA1丙氨酸（产朊假丝酵母）的解离常数估计为3.6×10^(-7) M，苯丙氨酰-tRNA苯丙氨酸（大肠杆菌）为7.9×10^(-8) M，起始甲硫氨酰-tRNAfMet（大肠杆菌）为8.1×10^(-7) M，甘氨酰-tRNA1甘氨酸（表皮葡萄球菌）为5.4×10^(-6) M。此外，通过使用相对大量的EF-Tu:GTP，即使采用凝胶过滤法，我们也能够检测到起始甲硫氨酰-tRNAfMet和甘氨酰-tRNA1甘氨酸与EF-Tu:GTP形成的三元复合物。
