Protein Synthesis in Brine Shrimp Embryos

We have purified from the ribosomal wash of dormant and developing embryos of Artemia two proteins, Co‐eIF‐2(A) and Co‐eIF‐2, (B). These factors are essential for ternary complex formation and binding of [ 35 S]‐Met‐tRNA f to 40‐S ribosomal subunits with 15–30 μg eIF‐2/ml of reaction mixture. On polyacrylamide gel electrophoresis in dodecylsulfate, Co‐eIF‐2(A) is composed of a single polypeptide of M r 65000, whereas Co‐eIF‐2(B) contains polypeptides of M r 105000 and 112000. Co‐eIF‐2(A) is sensitive to 4.5 μM aurintricarboxylic acid but Co‐eIF‐2(B) requires approximately 15 μM aurintricarboxylic acid to give 50% inhibiton of ternary complex formation. The stimulatory activity of both actors is abolished by pretreatment of the proteins with N ‐ethylmaleimide. Artemia eIF‐2 rapidly binds [ 3 H]GDP or [ 3 H]GTP and at 15°C the initiation factor rapidly equilibrates bound nucleotides with free GDP or GTP. Both Co‐eIF‐2(A) and Co‐eIF‐2(B) have no effect on the exchange or the amount of nucleotide bound. The small subunit ( M r 43000) of Artemia eIF‐2 is phosphorylated in the presence of the rabbit reticulocyte heme‐repressible kinase. Tryptic digestion of [ 32 P]phosphorylated eIF‐2 produces a single major phosphopeptide and several minor ones. Acid hydrolysis of these phosphopeptides, as well as of [ 32 P]phosphorylated eIF‐2, demonstrates that the radioactivity is predominantly associated with phosphoserine. Phosphorylated Artemia eIF‐2 is active in ternary complex formation, in AUG‐dependent binding of [ 35 S]Met‐tRNA f to 40‐S ribosomal subunits and in cell‐free protein synthesis. Both Co‐eIF‐2(A) and Co‐eIF‐2(B) stimulate ternary complex formation with phosphorylated eIF‐2. A kinase that phosphorylates the small subunit of eIF‐2 is present in the postribosomal supernatant as well as in the ribosomal wash of developing Artemia embryos.