Studies on the high molecular weight form of polypeptide chain elongation factor-1 from pig liver. II. Interaction with guanine nucleotides and aminoacyl-tRNA.

The high molecular weight form of polypeptide chain elongation factor-1, or EF-1H, has been purified from pig liver to apparent homogeneity and shown to be a 1:1:1 complex of EF-1 alpha, EF-1 beta, and EF-1 gamma, namely, EF-1 alpha beta gamma (Hattori, S. and Iwasaki, K. (1980) J. Biochem. 88, 725-736). Therefore, its enzymatic properties were investigated in detail. The effects of various components such as NH4Cl, Mg(CH3COO)2, guanine nucleotides and Phe-tRNA on its heat stability were investigated and it was found that its properties were very close to those of the aminoacyl-tRNA binding enzyme from rabbit reticulocytes (McKeehan, W.L. and Hardesty, B. (1969) J. Biol. Chem. 244, 4330-4339). Furthermore, it was reported that EF-1 alpha beta gamma seemed to dissociate into EF-1 alpha and EF-1 beta gamma when guanine nucleotides were present, and also to form a complex of EF-1 alpha . GTP . [14C]Phe-tRNA when both GTP and [14C]Phe-tRNA were present. On the contrary, however, when the interaction between EF-1 alpha beta gamma and guanine nucleotides was analyzed by gel filtration at 4 degrees C, the dissociation of EF-1 alpha beta gamma could not be detected, instead the formation of binary complexes containing EF-1 alpha beta gamma and guanine nucleotides was observed. The dissociation constants of the EF-1 alpha beta gamma . GDP and EF-1 alpha beta gamma . GTP complexes were estimated to be 6.8 x 10(-6) M and 7.3 x 10(-6) M, respectively, and the number of binding sites per molecule of EF-1 alpha beta gamma for these nucleotides was estimated to be one. Little, if any, interaction was detected between EF-1 alpha beta gamma and aminoacyl-tRNA even in the presence of GTP. The controversial results obtained by different methods as described above seemed to be compatible with each other, if one assumes that the dissociation constant for the conversion of EF-1 alpha beta gamma into EF-1 alpha and EF-1 beta gamma increased with the increase in temperature. When gel filtration of EF-1 alpha beta gamma was carried out with a solution containing both GTP and [14C]Phe-tRNA, the formation of a ternary complex containing EF-1 alpha, GTP and [14C]Phe-tRNA was detected at 4 degrees C, although the amount was very small. From these results, we assumed that EF-1 alpha beta gamma interacts with GTP and aminoacyl-tRNA (aa-tRNA) according to the following sequences; EF-1 alpha beta gamma + GTP in equilibrium with EF-1 alpha beta gamma . GTP EF-1 alpha beta gamma . GTP in equilibrium with EF-1 alpha . GTP + EF-1 beta gamma EF-1 alpha . GTP + aa-tRNA in equilibrium with EF-1 alpha . GTP . aa-tRNA.