Protein synthesis in rabbit reticulocytes. Purification and characterization of a double-stranded RNA-dependent protein synthesis inhibitor from reticulocyte lysates.
Reticulocyte lysates contain a latent form of eukaryotic peptide chain initiation factor 2 (eIF-2) kinase (dsI) which becomes activated in the presence of double-stranded RNA and ATP and inhibits protein synthesis. The latent form of dsI has been partially purified from reticulocyte ribosomal salt wash. The purified dsI has been activated by incubation in the presence of poly(rI).poly(rC) and [gamma 32P]ATP and the activated dsI has been further purified to near homogeneity. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified [32P]dsI shows an intensely staining 67,000-dalton polypeptide band which corresponds to a single 67,000-dalton radioactive band. During Sephadex (G-200) gel filtration, both the latent form of dsI and the activated dsI elute similarly with a peak corresponding to a molecular weight of 67,000. Purified dsI phosphorylates the 38,000-dalton subunit of eIF-2 and, under conditions of eIF-2 phosphorylation, dsI strongly inhibits AUG-dependent Met-tRNAf binding to 40 S ribosomes. Also, in partial reactions, eIF-2 alpha (P) formed by phosphorylation of eIF-2 using dsI and ATP, is not recognized by two eIF-2 ancillary factors, Co-eIF-2B and Co-eIF-2C. These results are similar to those reported previously for the heme-regulated eIF-2 kinase (Das, A., Ralston, R. O., Grace, M., Roy, R., Ghosh-Dastidar, P., Das H. K., Yaghmai, B., Palmieri, S., and Gupta, N. K. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,5076-5079) and suggest that dsI, like the heme-regulated eIF-2 kinase phosphorylates eIF-2 and eIF-2 alpha (P) thus formed, in both cases, is not recognized by Co-eIF-2B and Co-eIF-2C, and is inactive in some step(s) of Met-tRNAf.40 S initiation complex formation.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis*; D004789:Enzyme Activation / Q000187:drug effects; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000494:pharmacology*; D011506:Proteins / Q000037:antagonists & inhibitors*; D012330:RNA, Double-Stranded / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis*; D012270:Ribosomes / Q000032:analysis; D019892:eIF-2 Kinase
D001798:Blood Proteins; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D010448:Peptide Initiation Factors; D011506:Proteins; D012330:RNA, Double-Stranded; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D019892:eIF-2 Kinase