A Rapid Assay for the Initiation of Protein Synthesis in Extracts of Animal Cells

A rapid method for the quantitative and qualitative measurement of the initiation of protein synthesis in mammalian cell‐free extracts is described. Use of the antibiotic sparsomycin, which inhibits chain elongation but has little effect on initiation, results in the accumulation of short initiation peptides during protein synthesis in vitro. The initiation peptides remain bound to tRNA but are not attacked by the putative aminopeptidase responsible for the removal of N‐terminal methionine from nascent polypeptides. After electrophoresis and autoradiography such initiation peptides labelled with [ 35 S] methionine yield a specific fingerprint for each site at which initiation has occurred. The optimal conditions for cell‐free incubation, isolation and characterisation of initiation peptides are described.