Purification and characterization of initiation factor IF-E3 from rabbit reticulocytes.
Initiation factor IF-E3 from rabbit reticulocytes was isolated from a high salf extract of ribosomes prepared according to the procedure of Schreier and Staehelin (J. Mol9 Biol, 73, 329-349, 1973). The factor was highly purified from the crude extract by ammonium sulfate fractionation, sucrose gradient centrifugation, salf gradient elution from DEAE-cellulose and phosphocellulose columns, and glycerol gradient centrifugation. IF-E3 stimulated cell-free protein synthesis dependent on an exogenous globin mRNA fraction 4- to 5-fold. The factor under nondenaturing conditions behaved as a large multipolypeptide complex, but was separated into 11 major protein components by two-dimensional polyacrylamide gel electrophoresis with urea and sodium dodecyl sulfate. The stoichiometry and molecular weights (range: 28,000-140,000) of the IF-E3 proteins were determined. None of the components corresponded to ribosomal proteins found in high salt-washed ribosomes. 14CH3-IF-E3 was prepared by reductive alkylation without detectable loss of its initiation factor activity, and bound stoichiometrically to 40S ribosomal subunits, but not to 60S or 80S ribosomes. 14CH3-IF-E3 isolated from the 40S complex contained only nine of the 11 original protein components.
D000818:Animals; D001665:Binding Sites; D008722:Methods; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000032:analysis / Q000302:isolation & purification*; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis*; D012269:Ribosomal Proteins / Q000032:analysis; D012270:Ribosomes / Q000378:metabolism
D010448:Peptide Initiation Factors; D012269:Ribosomal Proteins