Studies on the High Molecular Weight Form of Polypeptide Chain Elongation Factor-1 from Pig Liver

Abstract

The high molecular weight form of the polypeptide chain elongation factor-1 (EF-1H) from pig liver was purified to an apparently homogeneous state. The procedure included aqueous two-phase separation, ammonium sulfate fractionation, four successive column chromatographies on Sephadex G-200, CM-Sephadex C-50, hydroxylapatite and octyl-Sepharose CL-4B, and glycerol density gradient centrifugation. By this procedure, the specific activity of EF-1H increased about 60-fold with a recovery of the total activity of 6%, as compared to the material obtained by aqueous two-phase separation followed by ammonium sulfate fractionation. Purified EF-1H was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions and glycerol gradient centrifugation, revealing that it is apparently homogeneous and has both EF-1 alpha and EF-1 beta gamma activities. The subunit structure of EF-1H was investigated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate as well as two-dimensional gel electrophoresis, which indicated that EF-1H consists of three different subunits, i.e., EF-1 alpha, EF-1 beta, and EF-1 gamma, in an equimolar ratio. The Stokes radius and the sedimentation coefficient of EF-1H were estimated to be 5.9 nm and 6.2S, respectively, from which the molecular weight of EF-1H was calculated to be about 157,000. This value is close to the sum of the molecular weights of EF-1 alpha (53,000), EF-1 beta (30,000), and EF-1 gamma (53,000). Thus, we concluded that EF-1H is a monomer of a 1:1:1 complex of EF-1 alpha, EF-1 beta, and EF-1 gamma, or EF-1 alpha beta gamma and its molecular weight is 136,000. This conclusion was further confirmed by measuring the amounts of EF-1 alpha and EF-1 beta gamma in EF-1H by enzymatic assays. The amino acid composition of EF-1H was determined, and the results revealed that there was a close resemblance between the values of EF-1H and those of EF-1 alpha beta gamma, which were calculated from the reported values for EF-1 alpha, EF-1 beta, and EF-1 gamma.