Purification and Properties of Polypeptide Chain Elongation Factor-1βγ from Pig Liver

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Abstract
We have previously shown that eukaryotic polypeptide chain elongation factor 1 (EF-1) from pig liver could be resolved into two complementary factors, EF-1α and EF-1β, of which the former corresponds to one of the bacterial elongation factors, EF-Tu. A brief description of the purification of EF-1β has also been presented (Iwasaki, K., Motoyoshi, K., Nagata, S., & Kaziro, Y. (1976) J. Biol. Chem. 251, 1843). This paper describes the purification procedure of EF-1β in detail as well as some properties of the purified factor, which was renamed EF-1βγ. The purification procedure includes an aqueous two-phase separation of 15,000 × g supernatant of pig liver homogenate, ammonium sulfate fractionation, treatment with sodium cholate and two successive column chromatographies on diethylaminoethyl-Sephadex A-50. By this procedure, EF-1βγ was purified about 40-fold starting from the material obtained after cholate treatment with a recovery of 20%. The purified EF-1βγ appeared to be homogeneous as judged by polyacrylamide gel electrophoresis. It had a molecular weight of about 90,000, and consisted of two unequal subunits of molecular weights of 55,000 and 30,000. The purified EF-1βγ stimulated three reactions: i) polymerization of phenylalanine dependent on poly(U) in the presence of both EF-1α a and EF-2, ii) EF-1α-dependent binding of phenylalanyl-tRNA to ribosomes in the presence of GTP but not in the presence of guanyl-5′-yl methylenediphosphonate, and iii) the exchange of GDP bound to EF-1α with exogenous GTP. These results strongly indicate that the function of EF-1βγ is to stimulte the conversion of EF-1α·GDP to EF-1αGTP, which results in the rapid recycling of EF-1α, and is, at least in part, similar to that of bacterial EF-Ts.