Purification and characterization of protein synthesis initiation factors eIF-1, eIF-4C, eIF-4D, and eIF-5 from rabbit reticulocytes.

Protein synthesis initiation factors eIF-1, eIF-4C, eIF4D, and eIF-5 were isolated and purified from rabbit reticulocyte lysates. Crude initiation factors from the high salt ribosomal extract were separated by ammonium sulfate precipitation into Fraction A (0 to 40% saturation) and Fraction BC (40 to 70% saturation). Fraction BC was separated by gel filtration on Sephadex G-100 into two fractions: one containing eIF-2 and eIF-5 and a second containing eIF-1, eIF-4A, eIF-4C, and eIF-4D. Initiation factor eIF-5 was separated from eIF-2 on phosphocellulose and further purified on DEAE-cellulose and Sephadex G200. eIF-5 has a molecular weight of 150,000 and was obtained 75 to 80% pure. Initiation factor eIF-4C was separated from eIF-1 and eIF-4D on phosphocellulose. eIF4C has a molecular weight of 17,500 and was obtained 90% pure. eIF-1 and eIF-4D were separated on DEAE-cellulose, and eIF-1 was further purified on Sephadex G-75. eIF-1 and eIF-4D have molecular weights of 15,000 and 16,500, respectively, and were obtained 80 to 85% pure. Each of the purified factors stimulated globin or methionylpuromycin synthesis, or both. The factors were radioactively labeled with 14C-methyl groups by reductive alkylation; eIF-4C, eIF-4D, and eIF-5 (when alkylated to a limited extent) retain their biological activities while modification of eIF-1 results in loss of activity. Two-dimensional urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the four initiation factors are different from the polypeptides of any other initiation factor or any proteins present in ribosomal subunits.