PROTEIN-SYNTHESIS IN RABBIT RETICULOCYTES - PURIFICATION AND PROPERTIES OF AN MR 80,000 POLYPEPTIDE (CO-EIF-2A80) WITH CO-EIF-2A ACTIVITY

  • 1 January 1985
    • journal article
    • research article
    • Vol. 260  (11) , 6945-6949
Abstract
The high MW protein complex, Co-eIF-2 [eukaryotic initiation factor 2 complex], contains both Co-eIF-2A and Co-eIF-2C activities. Co-eIF-2A stimulated Met-tRNAf binding to eukaryotic initiation factor-2 (eIF-2) both in the presence and absence of Mg2+. Co-eIF-2C stimulates Met-tRNAf binding to eIF-2 in the presence of Mg2+ by relieving Mg2+ inhibition of ternary complex formation from eIF-2. Co-eIF-2 protein complex contains several polypeptides including MW 80,000 and 50,000 polypeptides. Three polypeptides (MW 80,000, 50,000 and 25,000) are present in 0.5 M KCl ribosomal salt wash and each possesses Co-eIF-2A activity. MW 80,000 polypeptide (Co-eIF-2A80) has been purified to homogeneity and its properties studied. Co-eIF-2A80 stimualted Met-tRNAf binding to eIF-2 and the complex formed was resistant to aurintricarboxylic acid. Co-eIF-2A80 activity was N-ethylmaleimide-resistant and heat-labile; it was destroyed by heating at 55.degree. C for 4 min. Antibodies prepared against homogeneous Co-eIF-2A80 strongly inhibited protein synthesis is reticulocyte lysates and, also, eIF-2 and Co-eIF-2 promoted Met-tRNAf binding to 40 S ribosomes. Inhibition of protein synthesis in reticulocyte lysates was overcome by preincubation of anti-Co-eIF-2A80 with homogeneous Co-eIF-2A80 and was partially overcome by similar preincubation with Co-eIF-2. Upon limited digestion with Staphylococcus aureus V8 protease, the homogeneous Co-eIF-2A80 gave 2 major polypeptide fragments (MW 50,000 and 25,000). Upon similar treatment, an MW 80,000 polypeptide band isolated from the sodium dodecyl sulfate-gel of the Co-eIF-2 protein complex gave 4 major polypeptide fragments, and 2 of these fragments (MW 50,000 and 25,000) were similar to those given by Co-eIF-2A80, indicating that this MW 80,000 polypeptide band contains the Co-eIF-2A80 component. Co-eIF-2A80 is a component of Co-eIF-2 and is also essential for Co-eIF-2 activity and overall peptide chain initiation.

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