Purification of eukaryotic initiation factors eIF-2, eIF-2B and eIF-2 alpha kinase from bovine liver.

Abstract

Eukaryotic initiation factors 2 and 2B (eIF-2; eIF-2B) are components of the rate-limiting step in the initiation of eukaryotic protein synthesis and are involved in the regulation of this process. When the alpha-subunit of eIF-2 is phosphorylated by an eIF-2 alpha kinase, the phosphorylated eIF-2 alpha (eIF-2 alpha(P)) binds tightly to eIF-2B and prevents the recycling of eIF-2.GDP to eIF-2.GTP which is required for sustained initiation of protein synthesis. The minute quantities of these proteins which are present in rat liver and muscle cytosol along with hundreds of other proteins has hindered purification efforts, as well as structure:function and regulatory studies. Therefore, procedures were developed for the simultaneous purification of eIF-2, eIF-2B and eIF-2 alpha kinase from kilogram quantities of fresh bovine liver. Briefly, the 0-45% ammonium sulfate precipitate of the 200,000 x g supernatant was solubilized and chromatographed on DEAE-cellulose, heparin-agarose, Mono Q, Mono S, and Superose columns. The availability of purified quantities of these factors will be useful for investigations of molecular mechanisms of action and antibody production.