Purification and properties of eukaryotic initiation factor 2 and its ancillary protein factor (Co-eIF-2A) from yeast Saccharomyces cerevisiae.
Two peptide chain initiation factor activities, eIF-2y and Co-eIF-2A20y, were purified from the high speed supernatant fraction of the yeast Saccharomyces cerevisiae and their properties were studied. 1) In sodium dodecyl sulfate-polyacrylamide gels, purified eIF-2y showed two major polypeptide bands corresponding to molecular weights of 54,000 and 36,000. The Mr 54,000 band was significantly more intense than the Mr 36,000 band, indicating the possible presence of two polypeptides of equal molecular weight in this band. The molecular weight of eIF-2y, determined using a density gradient centrifugation method, was approximately 140,000. 2) In sodium dodecyl sulfate-polyacrylamide gel, purified Co-eIF-2A20y showed a single polypeptide band corresponding to a molecular weight of 20,000. A similar molecular weight for Co-eIF-2A20y was also found using a density gradient centrifugation method. 3) In partial reactions, eIF-2y bound Met-tRNAf in the presence of Mg2+. The reaction required GTP. Co-eIF-2A20y stimulated Met-tRNAf binding to eIF-2y (2-3-fold) and also rendered the complex stable to 3 X 10(-5) M aurintricarboxylic acid. 4) This Co-eIF-2A20y activity was heat-labile and N-ethylmaleimide-insensitive. 5) Antibodies were prepared by injecting rabbits with homogeneous Co-eIF-2A20y. Such anti-Co-eIF-2A20y inhibited (60%) protein synthesis in a yeast cell-free protein synthesizing system and completely blocked Co-eIF-2A20y stimulation of Met-tRNAf. 40 S initiation complex formation. Protein synthesis inhibition by anti-Co-eIF-2A20y was almost completely reversed by preincubation of the antibodies specifically with homogeneous Co-eIF-2A20y.
D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D006358:Hot Temperature; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D012441:Saccharomyces cerevisiae / Q000032:analysis*
D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D010448:Peptide Initiation Factors; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D005033:Ethylmaleimide