PURIFICATION OF EUKARYOTIC INITIATION FACTOR-EIF-2, FACTOR-EIF-2B ANDFACTOR-EIF-2-ALPHA-KINASE FROM BOVINE LIVER

Eukaryotic initiation factors 2 and 2B (eIF-2; eIF-2B) are components of the rate-limiting step in the initiation of eukaryotic protein synt hesis and are involved in the regulation of this process. When the alp ha-subunit of eIF-2 is phosphorylated by an eIF-2alpha kinase, the pho sphorylated eIF-2alpha (eIF-2alpha(P)) binds tightly to eIF-2B and pre vents the recycling of eIF-2.GDP to eIF-2.GTP which is required for su stained initiation of protein synthesis. The minute quantities of thes e proteins which are present in rat liver and muscle cytosol along wit h hundreds of other proteins has hindered purification efforts, as wel l as structure:function and regulatory studies. Therefore, procedures were developed for the simultaneous purification of eIF-2, eIF-2B and eIF-2alpha kinase from kilogram quantities of fresh bovine liver. Brie fly, the 0-45% ammonium sulfate precipitate of the 200,000 x g superna tant was solubilized and chromatographed on DEAE-cellulose, heparin-ag arose, Mono Q, Mono S, and Superose columns. The availability of purif ied quantities of these factors will be useful for investigations of m olecular mechanisms of action and antibody production.