The purification and characterization of multiple forms of protein synthesis eukaryotic initiation factors 2, 3, and 5 from rabbit reticulocytes.
Eukaryotic initiation factors 2, 3, and 5 (eIF-2, eIF-3, and eIF-5) each were purified or fractionated into a number of forms which differ in protein mass or composition. The various factor polypeptides were analyzed by partial protease digestion and the fragmentation patterns were compared to identify proteins related in primary structure. The digestion method was made more sensitive by prior radiochemical labeling of the proteins by iodination with 125I. At least four molecular weight forms of eIF-5 were found which ranged from 168,000 to 128,000. The lower molecular weight forms, nevertheless, were biologically active in in vitro assays. Analysis of a variety of eIF-3 preparations indicates that the presence or abundance of especially the higher molecular weight subunits varies considerably. Many of these components are related to one another in primary structure; five of the subunits greater than Mr = 90,000 appear to be derived from the 210 kilodalton subunit by limited proteolysis. Analysis of eIF-2 polypeptides shows that the alpha, beta, and gamma subunits give distinctively different partial protease digestion patterns and that some preparations contain a minor component (gamma') related to the gamma subunit which could be misidentified as the beta subunit. The results indicate that initiation factors may be cleaved into different active forms by proteases acting either in the intact cell or during the factor isolation and purification. Addition of the protease inhibitor, phenylmethanesulfonyl fluoride, prevents some, but not all of the proteolysis.
D000818:Animals; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects; D010448:Peptide Initiation Factors / Q000097:blood / Q000302:isolation & purification*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D010664:Phenylmethylsulfonyl Fluoride