Purification of Eukaryotic Initiation Factors elF-2, eIF-2B and elF-2a Kinase from Bovine Liver

ABSTRACT

Eukaryotic initiation factors 2 and 2B (elF-2; elF-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 a-subunit of elF-2 is phosphorylated by an elF-2oc kinase, the phosphorylated elF-2a (elF- 2a(P)) binds tightly to elF-2B and prevents the recycling of elF-2#x00AB;GDP to elF-2#x00AB;GTP which is required for sustained initiation of protein synthesis. he 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 elF-2, elF-2B and elF-2a kinase from kilogram quantities of fresh bovine liver. Briefly, the 0-45% ammonium sulfate precipitate of the 200,000 x gsupernatant 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.