Identification of initiation factors and ribosome-associated phosphoproteins by two-dimensional polyacrylamide gel electrophoresis.
A two-dimensional polyacrylamide gel electrophoresis procedure has been used to identify initiation factors rapidly in the high-salt-wash fraction from reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ATP/GTP/Pi exchange system is described which has been used to maintain [gamma-32P]ATP and [gamma-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex have been identified using a combination of the two procedures. The salt-wash fraction contains eight major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins are observed to comigrate with two of the three subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf onto the 40-S subunit and promoting dissociation of 80-S ribosomes. eIF-4B, one of the proteins involved in binding mRNA to 40-S subunits is also phosphorylated. The remainder of phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and have not been identified further. Two of the six phosphoproteins associated with the salt-washed ribosomes comigrate with ribosomal proteins; one is the major phosphorylated protein in 40-S ribosomal subunits, the other is an acidic protein.
D000818:Animals; D004586:Electrophoresis; D004589:Electrophoresis, Disc; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000032:analysis*; D010750:Phosphoproteins / Q000097:blood*; D010766:Phosphorylation; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis; D012269:Ribosomal Proteins / Q000032:analysis*; D012270:Ribosomes / Q000032:analysis
D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D012269:Ribosomal Proteins