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By means of affinity chromatography of lysates from Krebs I1 ascites cells and rabbit reticulocytes on Sepharose-heparin an active fraction of initiation factors has been obtained.The fraction is eluted from the column at 350 mM KC1 using a linear gradient and displays a number of activities, i. e. binding of Met-tRNAy" to form a ternary complex with GTP ; transferring this complex to 40-S subunits in an A-U-G-independent step and finally coupling of the 40-S initiation complex to the 60-S subunit, a reaction which is completely A-U-G-dependent. Moreover, MettRNA is bound into the P-site as is indicated by its puromycin sensitivity. The method is very suitable for large-scale preparation. Further purification and characterization of the factors have been carried out on DEAE-cellulose and phosphocellulose columns.Evidence is presented that the polysomes present in a lysate that has been passed through the Sepharose-heparin column can only complete their nascent chains, initiation of new polypeptides is completely dependent on addition of initiation factors.

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SummaryThe eukaryotic initiation factors eIF-4A and eIF-4D are almost exclusively found in the supernatant of a reticulocyte lysate. The basic steps of general purification schemes failed because these factors do not bind to Sepharoseheparin or, in the case of eIF-4A, to phosphocellulose and a new procedure had to be devised. Cibacron Blue F3G-A proved to be a successful alternative in the purification of these factors from the excessive load of proteins found in the supernatant.