Open AccessInitiation of Protein Synthesis in Eukaryotes
Author(s) -
MAST Cor,
THOMAS Adri,
GOUMANS Hans,
AMESZ Hans,
VOORMA Harry O.
Publication year - 1977
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1977.tb11548.x
Subject(s) - sepharose , puromycin , elution , ternary complex , chromatography , chemistry , affinity chromatography , gtp' , protein subunit , biochemistry , lysis , protein biosynthesis , enzyme , gene
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‐tRNA Met f 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, Met‐tRNA 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.