Initiation of protein synthesis in eukaryotes. Binding to Sepharose-heparin and partial purification of initiation factors from Krebs II ascites cells.
By means of affinity chromatography of lysates from Krebs II 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 KCl using a linear gradient and displays a number of activities, i.e. binding of Met-tRNAfMet 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.
D000818:Animals; D001665:Binding Sites; D002287:Carcinoma, Krebs 2 / Q000378:metabolism*; D002846:Chromatography, Affinity; D006160:Guanosine Triphosphate / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D008715:Methionine; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects; D010448:Peptide Initiation Factors / Q000302:isolation & purification; D011189:Potassium Chloride / Q000494:pharmacology; D011691:Puromycin / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D010448:Peptide Initiation Factors; D011691:Puromycin; D011189:Potassium Chloride; D006160:Guanosine Triphosphate; D006493:Heparin; D012343:RNA, Transfer; D008715:Methionine