Isolation and purification of hen oviduct protein synthesis initiation factors A2A and A2B

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Abstract
Two initiation factors, IF-A2A and IF-A2B, required for protein synthesis in a fractionated system were isolated from hen oviduct. These factors were obtained from a 0.5 M KCl extraction of a nuclear-microsomal fraction of the oviduct. The crude extract inhibited protein synthesis, but, following DEAE-cellulose chromatography, activity was detected. Sephadex G-200 chromatography separated the activity into 2 active fractions, A2A and A2B. These factors were characterized with respect to their activities in polyphenylalanine polymerization at a low Mg2+ concentration, AUG and ATP dependent Met-tRNAf binding to 40S ribosomal subunits, the hydrolysis of GTP and natural mRNA dependent protein synthesis. In all of these systems A2A and A2B were able to substitute for rabbit reticulocyte M2A and M2B. The molecular weights of A2A and A2B were estimated by gel filtration chromatography to be 280,000 and 23,000, respectively. A2A sedimentation analysis on sucrose gradients showed a sedimentation coefficient of 5.2 S. Combining these data, the molecular weights of A2A was calculated to be 125,000. These values are similar to those for corresponding reticulocyte proteins. In the presence of added ovalbumin mRNA, A2A and A2B stimulated protein synthesis on non-salt-washed hen oviduct and rabbit reticulocyte polysomes. A2A and A2B dependent synthesis of ovalbumin also to occur on reticulocyte polysomes programmed with ovalbumin mRNA. The conclusion that these factors are initiation factors for protein synthesis and not ribosomal structural proteins was supported.