Purification and characterization of initiation factor IF-E3 from rabbit reticulocytes.

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Abstract
Initiation factor IF-E3 from rabbit reticulocytes was isolated from a high salt extract of ribosomes prepared according to the procedure of Schreier and Staehelin. The factor was highly purified from the crude extract by ammonium sulfate fractionation, sucrose gradient centrifugation, salt 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 2-dimensional polyacrylamide gel electrophoresis with urea and sodium dodecyl sulfate. The stoichiometry and MW (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 9 of the 11 original protein components.