Abstract
Initiation factor IF-E2 was isolated from rabbit reticulocytes and purified 120-fold to near homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and phosphocellulose, and, when suitable, by sucrose density gradient centrifugation. The factor is a complex protein containing three nonidentical polypeptides of molecular weight 57,000, 52,000, and 36,000. It behaves as a complex throughout its purification and during polyacrylamide gel electrophoresis in nondenaturing buffer but its thress components are readily separated by electrophoresis in denaturing buffers. None of its components corresponds to any of the polypeptides of the other initiation factors or to any proteins of ribosomes washed in buffers containing a high salf concentration. A stoichiometric ratio of 1:1:1 was determined for the three polypeptides; based on the assumption of one copy each per complex, the calculated factor molecular weight is 145,000, a value in agreement with the measured value of 160,000. Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase also isolated from rabbit reticulocytes. Neither procedure causes a measurable change in the ability of the factor to form a ternary complex with GTP and the initiator methionyl-tRNA. 5'-Guanylyl-methylenediphosphonate may substitute for GTP, but only at relatively high concentrations. The binding of labeled initiation factor IF-E2 and methionyl-tRNA to the 40 S ribosomal subunit was studied by sucrose density gradient centrifugation. Appreciable binding of the factor is seen only when all three components of the ternary complex are included in the reaction mixture. The binding of either the factor or methionyl-tRNA was not stimulated by the addition of globin messenger RNA and initiation factor IF-E3. It was shown that all three polypeptide components of initiation factor IF-E2 are bound to these nascent initiation complexes.
Highlights
Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase isolated from rabbit reticulocytes
IF-E2 (10 pg, following chromatography on phosphocellulose) was analyzed by SDS-PAGE electrophoresis as described in Fig. 2 and the stained gel was scanned at 600 nm in a Gilford 2400 spectrophotometer with a linear transport attachment
IF-E2 was purified from the high salt ribosomal wash from rabbit reticulocytes
Summary
Crude initiation factors from rabbit reticulocvtes, rat liver ribosomes, rat liver pH 5 enzyme fraction, and globin mRNA were prepared as described previously [12]. Unfractionated tRNA was isolated from rabbit live; [13] and-the initiator tRNA was charged with L3Hlmethionine (specific activity, 3.7 Cilmmol) by using an Escherichia coli synthetase preparation according to the. Saturation) [12], obtained from the reticulocytes of 60 rabbits, was dialyzed against 20 rnM Tris/HCl, pH 7.6, 7 rnM 2-mercaptoethanol, and 5% glycerol (Buffer A) containing 100 rnM KCl. The solution (9 ml; 80 mg of protein) was diluted with 1 volume of water and applied to a DEAE-cellulose column (Whatman DE32; 0.9 x 30 cm) equilibrated in Buffer A and 50 rnM KCl. The adsorbed protein was eluted with a 200-ml linear gradient of 50 to 300 rnM KC1 in Buffer A, and fractions were assayed for IF-E2 activity as described below. The data were analyzed by the method of Hedrick and Smith [21]
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