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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. [ABSTRACT FROM AUTHOR]

1. Decapped tobacco mosaic virus (TMV) RNA and rabbit globin mRNA were prepared by enzymic treatment of RNAs with nucleotide pyrophosphatase purified from potato. The extent of removal of 5'-terminal 7-methylguanosine 5'-monophosphate (m GMP) from TMV RNA was at least 97% as estimated by labeling of the 5' termini in vitro with S-adenosyl[methyl-³H]methionine catalysed by vaccinia virus methyltransferases. 2. The effect of enzymic decapping was compared with the effect of cap analogs on mRNAs translation in a nuclease-treated rabbit reticulocyte lysate and in a wheat germ extract. When translation was studied at low K+ concentration, little or no dependence on 5'-terminal 7-methylguanosine was found with either cell-free system. The importance of the 5'-terminal cap for the efficient translation of TMV RNA and globin mRNA increased as the concentration of K+ in a protein-synthesis system was raised. In a reticulocyte lysate analogs and enzymic decapping had a similar effect on translation. In a wheat germ extract, mRNA decapping resulted in a more pronounced decrease of mRNA activity, presumably due to the increased susceptibility of decapped mRNAs to the nucleases present in this protein synthesis system. 3. The requirement for a 5'-terminal cap was similar for the synthesis of 130 000-Mr and 165 000-Mr polypeptides coded by TMV RNA. This indicates that both proteins may be initiated at the common site close to the 5' terminus. [ABSTRACT FROM AUTHOR]