Purification of protein synthesis initiation factor eIF-3 from rat liver microsomes by affinity chromatography on rRNA-cellulose.
An efficient four-step procedure is described for preparing highly purified polypeptide chain initiation factor eIF-3 from rat liver microsomal saltwash. The method involves fractionation with ammonium sulfate between 25-40% saturation (0 degree C) followed by affinity chromatography on rRNA-cellulose, DEAE-cellulose chromatography and sucrose density gradient centrifugation, eIF-3 is eluted from the affinity column at a KCl concentration of 0.18 M. The purification is 10-times and the recovery of activity better than 85%. In the sucrose gradients, eIF-3, sediments as a 15 S particle indicating a total mass of 650 000 Da. The purified eIF-3 is highly active in stimulating globin synthesis in a fractionated translation system Factor eIF-3 contains eight subunits with molecular weights ranging from 40 000 to 110 000. Seven of the subunits are present in one copy per eIF-3, whereas the factor contains two copies of one subunit. The isoelectric points of the factor subunits range from 5.5 to 7.3 with most of the polypeptides being acidic.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D039621:Eukaryotic Initiation Factor-3; D007526:Isoelectric Point; D008862:Microsomes, Liver / Q000032:analysis*; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000378:metabolism; D051381:Rats
D039621:Eukaryotic Initiation Factor-3; D010448:Peptide Initiation Factors; D012335:RNA, Ribosomal