Initiation Factor eIF‐2 from Wheat Germ

Protein synthesis initiation factor eIF-2 was isolated and purified 4300-fold from wheat germ 23000 ×g supernatant. The procedure described resulted in a 70% pure factor and involves three chromatographic steps only: (a) on Sepharose-heparin, (b) on Sepharose–concanavalin-A and (c) on phosphocellulose. The Sepharose-concanavalin-A column was required for the removal of the bulk of the proteins present in the wheat germ supernatant, its use increased the speed of the procedure considerably. The factor consists of three non-identical subunits in a 1:1:1 stoichiometric ratio with apparent Mr of 56000, 41000 and 36000, which results in an Mr of 133000 for the undissociated factor. The subunits were found to be distinct from those of the eIF-2 from reticulocytes. The factor was tested in the various model reactions of the initiation process such as ternary complex formation with Met-tRNAfMet and GTP, methionyl-puromycin synthesis and in unfractionated wheat germ 23000 ×g supernatant and rabbit reticulocyte 30000 ×g supernatant systems. The results support the idea of an universal mode of action of initiation factors in eukaryotic cells: i.e. the wheat germ factor stimulated the above-mentioned systems in a manner similar to eIF-2 from rabbit reticulocytes. The two factors are fully exchangeable in ternary complex formation (both factors are approximately 30% active, reach maximum levels of [3H]Met-tRNAfMet binding at 0.6 μM GTP and show very little dependence on the Mg2+ concentration) and, provided that small amounts are used, in the assay for methionyl-puromycin and in the crude rabbit reticulocyte lysate. Both factor preparations act as a substrate for a cAMP-independent protein kinase from reticulocytes (hemin-regulated translational inhibitor) which is thought to be responsible for the shut-off of protein synthesis in hemin-deprived lysates from rabbit reticulocytes: the 41000-Mr subunit of wheat germ eIF-2 and the 38000-Mr, subunit of reticulocytes eIF-2 were stoichiometrically phosphorylated in vitro by this inhibitor in the presence of [γ-32P]ATP. Indeed, large amounts of the inhibitor inhibited protein synthesis in crude wheat germ extracts to some extent (25%). However, such an inhibition could not be overcome by the addition of purified eIF-2. Furthermore, when crude wheat germ extracts were incubated with [γ-32P]ATP, the eIF-2 subunit of Mr 36000 (but not the others) became phosphorylated, although no decrease in the rate of protein synthesis could be detected.