The isolation and characterization from rabbit reticulocytes of two forms of eukaryotic initiation factor 2 having different beta-polypeptides.
We have isolated from the high salt wash of rabbit reticulocyte ribosomes two forms of the polypeptide chain initiation factor 2 (eIF-2) which differ with respect to their beta-subunit, GDP content, and sensitivity to Mg2+ in ternary (eIF-2 X GTP X Met-tRNAf) and binary (eIF-2 X GDP) complex formation. The form of eIF-2 eluting first from a cation exchange (Mono S, Pharmacia) column has a beta-subunit of lower molecular weight (eIF-2(beta L] and a more acidic pI value than the form eluting at a higher salt concentration (eIF-2(beta H]. These two forms of eIF-2 beta-polypeptides are also detected in reticulocyte lysates when the proteins are resolved by two-dimensional isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting. The peptide mapping of the isolated beta-subunits after limited proteolysis by papain, pancreatic protease, alpha-chymotrypsin, or Staphylococcus aureus V8 protease further demonstrates that the two forms of beta-subunits are not the product of a non-specific proteolytic action that occurred during the purification procedure, but rather reflects the existence in vivo of both forms of eIF-2. The GDP content of eIF-2(beta L) and eIF-2(beta H) is approximately 0.85 and 0.22 mol of GDP/mol of eIF-2, respectively. The KD for GDP of eIF-2(beta L) was lower (2.2 X 10(-9) M) than that of eIF-2(beta H) (6.0 X 10(-8) M). In the presence of 1 mM Mg2+, the activities of eIF-2(beta L) and eIF-2(beta H) in forming a binary and a ternary complex are inhibited 90 and 25%, respectively. The extent of Mg2+ inhibition and its reversal by the guanine nucleotide exchange factor is directly proportional to the amount of GDP bound to eIF-2. No inhibition by Mg2+ is observed when eIF-2-bound GDP is removed by alkaline phosphatase. In the presence of the guanine nucleotide exchange factor, both forms of eIF-2 are equally active in ternary complex formation, and the complex formed is quantitatively transferred to 40 S ribosomal subunits.
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D000818:Animals; D001798:Blood Proteins; D002851:Chromatography, High Pressure Liquid; D015852:Eukaryotic Initiation Factor-2; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D010447:Peptide Hydrolases / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000097:blood / Q000302:isolation & purification*; D010455:Peptides / Q000097:blood*; D011506:Proteins / Q000302:isolation & purification*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis*
D001798:Blood Proteins; D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D010455:Peptides; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010447:Peptide Hydrolases