Protein synthesis in Drosophila melanogaster embryos. Purification and characterization of polypeptide chain-initiation factor 2.
Eukaryotic initiation factor 2 (elF-2) was purified from the high-salt wash fraction of Drosophila melanogaster embryos. This factor, with a molecular mass of about 90 kDa, consists of two subunits of 47 kDa and 39 kDa on dodecylsulfate/polyacrylamide gel electrophoresis. The 39-kDa subunit is phosphorylated by the hemin-controlled inhibitor of rabbit reticulocytes in a terminal fragment which can be cleaved by mild treatment with trypsin. Drosophila elF-2 is not a substrate for protein kinases capable of phosphorylating the beta subunit of elF-2 from rabbit reticulocytes. It is also shown that Drosophila elF-2 can form a ternary complex with GTP and Met-tRNAi, which can be efficiently transferred to 40S ribosomes in the presence of AUG and Mg2+. This factor is able to form a binary complex with GDP. Furthermore, purified elF-2 contains about 0.3 mol bound GDP/mol suggesting a high affinity of the factor for this nucleotide. Data supporting the notion that this affinity is increased in the presence of Mg2+, which impairs the GDP/GTP exchange on elF-2, are presented. The properties of Drosophila elF-2 suggest that this factor may be susceptible to regulation by a mechanism like that operating on rabbit reticulocyte elF-2.
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000032:analysis; D004331:Drosophila melanogaster / Q000196:embryology / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2; D006153:Guanosine Diphosphate / Q000032:analysis; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000302:isolation & purification*; D010449:Peptide Mapping; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification*; D012270:Ribosomes / Q000032:analysis
D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D011506:Proteins; D006153:Guanosine Diphosphate; D008274:Magnesium