Vicia sativaL. ‘Run-off’ and Purified Ribosomes: Polyphenylalanine Synthesis and Molecular Action of Ribosome-inactivating Proteins

A ‘run-off’ cell-free translation system (a 30000 × g supernatant; S 30) has been prepared from 4–5-d-old embryonic axes of Vicia sativa L., a plant lacking ribosome-inactivating protein activities which is very sensitive to certain RIPs but not to others. The system was able to generate a high rate of polyphenylalanine synthesis upon addition of polyuridylic acid. From this supernatant, purified ribosomes and a 100000 × g supernatant were prepared which were able to perform polyphenylalanine synthesis when mixed together (reconstituted system). The most important translation parameters were optimized in each case. Both the S 30 and the reconstituted system displayed differential sensitivities to certain RIPs. The purified RIP-inactivated ribosomes were able to release a 370 nucleotide rRNA fragment diagnostic for RIPs upon treatment of the isolated rRNA with acid aniline.