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

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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 chromatog-raphies 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-1n 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-lα and EF-lβγ 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-lα, EF-1β, and EF-lγ, 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-lα (53,000), EF-lβ (30,000), and EF-1γ-(53,000). Thus, we concluded that EF-1H is a monomer of a 1 :1 : 1 complex of EF-lα, EF-1β, and EF-lgama;, or EF-lαβγ and its molecular weight is 136,000. This conclusion was further confirmed by measuring the amounts of EF-lα and EF-1βγ 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-lαβγ, which were calculated from the reported values for EF-lα, EF-1β, and EF-1γ.

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