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

Abstract

Two initiation factors, IF-A2A and IF-A2B, required for protein synthesis in a fractionated system have been 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 two active frations, A2A and A2B. These factors have been characterized with respect to their activities in polyphenylalanine polymerization at a low Mg2+ concentration, AUG- and GTP-dependent Met-tRNAf binding to 40S ribosomal subunits, the hydrolysis of GTP, and natural messenger ribonucleic acid (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 have been estimated by gel filtration chromatography to be 280 000 and 23 000, respectively. Sedimentation analysis on sucrose gradients showed A2A to have a sedimentation coefficient of 5.2 S. Combining these data, the molecular weight of A2A was calculated to be 125 000. These values are similar to those for corresponding reticulocyte proteins. Finally, in the presence of added ovalbumin mRNA, A2A and A2B stimulated protein synthesis on non-salt-whashed hen oviduct and rabbit reticulocyte polysomes. Moreover, A2A- and A2B-dependent synthesis of ovalbumin was shown to occur on reticulocyte polysomes programmed with ovalbumin mRNA. This supports the conclusion that these factors are initiation factors for protein synthesis and not ribosomal structural proteins.

Partial purification and characterization of two hen oviduct protein synthesis initiation factors capable of initiation complex formation

“…The high speed supernatant was prepared from reticulocyte lysate by the method of Moldave et al (1971). Partially purified elongation factors (EF-1 and EF-2) were isolated from this supernatant as described previously (Hejtmancik & Comstock, 1976).…”

“…Isolation of Reticulocyte 40S Ribosomal Subunits. Active 40S ribosomal subunits were prepared by incubation with puromycin and 10-30% sucrose gradient centrifugation as previously described (Hejtmancik & Comstock, 1976).…”

Initiation of eukaryotic protein synthesis

“…The role of eIF-4D is obscure [26,84,85]. eIF4D stimulates the formation of the dipeptide-analogue methionyl-puromycin and the poly(U)-dependent polyphenylalanine synthesis [25,84,85].…”

“…eIF4D stimulates the formation of the dipeptide-analogue methionyl-puromycin and the poly(U)-dependent polyphenylalanine synthesis [25,84,85]. It slightly lowers the [Mg'+] for optimal protein synthesis [9], but does not affect the level of translation [9,25].…”

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“…The role of eIF-4D is obscure [26,84,85]. eIF4D stimulates the formation of the dipeptide-analogue methionyl-puromycin and the poly(U)-dependent polyphenylalanine synthesis [25,84,85]. It slightly lowers the [Mg'+] for optimal protein synthesis [9], but does not affect the level of translation [9,25].…”