Abstract
Eukaryotic polypeptide chain elongation factor-1α (EF-1α) has been purified from pig liver by steps including aqueous two-phase separation, ammonium sulfate fractionation, and three successive column chromatographies on CM-Sephadex, DEAE-Sephadex, and CM-Sephadex. On the last column chromatography on CM-Sephadex, EF-1α activity was eluted as four peaks. These peaks except the first one appear to be homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and isoelectric focusing in polyacrylamide gel. There was no difference between these four peaks in the activity to promote the binding of Phe-tRNA to ribosomes in the presence of guanyl-5'-yl methylenediphosphonate. They had similar molecular weights and similar amino acid compositions but different isoelectric points which varied from 9.3 to 9.9. The molecular weight of EF-1α was estimated to be 53, 000 and 61, 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by gel filtration on Sephadex G-150, respectively, indicating that EF-1α is composed of a single polypeptide. EF-1α appears to contain 6 half-cystine residues per molecule, at least one of which is essential for its activity. The effect of the concentration of NH4Cl on the dissociation constant of the binary complex containing EF-lα and guanine nucleotides (EF-1α•GDP or EF-1α•GTP) was extensively investigated, and it was found that the logarithm of the dissociation constant of EF-1α•GDP increased proportionally to the square root of the concentration of NF4Cl, while that of EF-1α•GTP decreased. Similar relationships were observed between the rate constant of dissociation of EF-1αGDP or EF-1αGTP and the concentration of NH4Cl. When the rate constant of association was calculated from these values, the logarithm of the constant of EF-1α•GDP and that of EF-1α•GTP found to decrease proportionally to the square root of the NH4Cl concentration.
