Purification of various forms of elongation factor 1 from rabbit reticulocytes.
Previous studies have indicated that the high-molecular-weight form of elongation factor 1 (EF-1H) contained four subunits (alpha, beta, gamma, and delta). Using the conventional methods of gel-filtration and ion-exchange chromatography, various forms of elongation factor 1 (EF-1 alpha, EF-1 beta delta, EF-1 beta gamma delta) have been purified from rabbit reticulocyte lysate. The procedure described allows one to purify these factors from a single batch of lysate in sufficient amounts for physical and biochemical studies. EF-1 alpha is a single polypeptide of Mr 52,000, and has an isoelectric point of 9.1. EF-1 beta delta and EF-1 beta gamma delta are composed of two and three nonidentical polypeptides, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both proteins can form stable aggregates in native conditions that can reach more than 2,000,000 Da. The isoelectric point for each polypeptide was determined; 5.8 for EF-1 beta, 5.5 for EF-1 gamma, and 4.8 for EF-1 delta. The activity of both proteins was compared on a molecular basis by their ability to stimulate EF-1 alpha in the poly(U)-directed synthesis of polyphenylalanine. On the basis of this assay EF-1 beta gamma delta is slightly more active than EF-1 beta delta. The similarity of the amino acid composition of EF-1 gamma and EF-1 delta and the molar ratio of alpha: beta: gamma: delta in EF-1H of approximately 1:1:0.5:0.5 have led to the conclusion that EF-1 delta is probably a breakdown product of EF-1 gamma, and that the native form of EF-1H probably contains only the alpha, beta, and gamma subunits.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008970:Molecular Weight; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000097:blood / Q000302:isolation & purification*; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis*
D000596:Amino Acids; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors