Protein synthesis in rabbit reticulocytes XXXI: Purification of Co-eIF-2C and studies of its roles in peptide chain initiation

Co-eIF-2C activity has been purified from high salt wash of reticulocyte ribosomes. The purified preparation is free from detectable levels of eIF-2, Co-eIF-2A, Co-eIF-2B and RF activities. The final step in the purification process involves use of a phosphocellulose chromatography and elution of Co-eIF-2C activity from the column with a buffer containing 2 M urea. This step results in complete removal of the contaminating Co-eIF-2B activity from the purified Co-eIF-2C preparation. Upon polyacrylamide gel electrophoresis, the final Co-eIF-2C preparation shows a single protein band under non-denaturing conditions. In the presence of SDS, the gel picture shows five prominent polypeptide bands (approximate mol. wt: 100,000; 67,000; 53,000; 45,000; and 40,000) and several faint bands. Purified Co-eIF-2C preparation strongly stimulates Met-tRNA_f·40S·AUG complex formation in the presence of eIF-2 and such stimulation is almost completely inhibited by HRI plus ATP. This study thus delineates the minimum factor requirements, namely, eIF-2 and Co-eIF-2C for formation of a stable Met-tRNA_f·40S·AUG complex.