Second phosphorylation site on alpha subunit of eIF-2 in rabbit reticulocyte lysate.
Eukaryotic protein synthesis initiation factor 2, eIF-2, was purified from either hemin-supplemented (translationally active) or hemin-deficient (translationally inactive) rabbit reticulocyte lysate under conditions chosen and demonstrated to preserve the in situ phosphorylation state. Direct analysis of the phosphate content of the alpha-subunit of eIF-2 was determined by chemical analysis of the isolated alpha-subunit or by a combination of vertical slab gel isoelectric focusing and immunoblotting. These results were compared with those obtained from an indirect analysis utilising the incorporation of [gamma-32P]ATP into eIF-2 by the heme-sensitive eIF-2 alpha-kinase. All three analyses demonstrate that the phosphorylation site specific for the heme-sensitive kinase is unoccupied in translationally active lysate and 25-30% occupied in translationally inhibited lysate. In addition, both direct analyses support the existence of a second phosphorylation site on the alpha-subunit, not regulated by hemin and distinct from that phosphorylated by the heme-sensitive kinase. Different reticulocyte lysate batches vary with respect to the activity of the kinase responsible for phosphorylation of the second site. Further investigations demonstrated that this kinase is a membrane-associated protein.
Department of Microbiology, University of Pittsburgh School of Medicine, Pennsylvania.
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D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D015852:Eukaryotic Initiation Factor-2; D006418:Heme / Q000494:pharmacology*; D007525:Isoelectric Focusing; D010448:Peptide Initiation Factors / Q000032:analysis / Q000378:metabolism*; D010710:Phosphates / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D010710:Phosphates; D011506:Proteins; D006418:Heme; D011494:Protein Kinases