Initiation and elongation of protein synthesis: Their relative rates and sensitivities to inhibition in Chinese hamster ovary cells determined by a modified Edman procedure

Abstract

The relative rates of initiation and elongation of protein synthesis are determined in exponentially growing cells from the incorporation of [ 35S]methionine into N-terminal and internal positions of growing peptide chains by a modified Edman degradation. Sequential samples of labeled cells are precipitated into filter paper supports with trichloroacetic acid. Large groups of samples can then be incubated in bulk with phenylisothiocyanate. Individual samples are treated with trifluoroacetic acid, and the derivatized N-terminal amino acids extracted from the filter paper with ethylene dichloride. The percentage of incorporation which is N-terminal varies with the purity of the [ 35S]methionine. Elevated temperature and hypertonicity, inhibitors of initiation, preferentially block incorporation into the N-terminal fraction (initiation) but allow continued incorporation into internal positions of previously initiated peptides. Puromycin inhibited incorporation into both fractions, as expected for an inhibitor of elongation.