Effects of nitrogen mustard on protein synthesis in a cell-free system from liver

Abstract

Nitrogen mustard (HN2), 1·10 −3 M, inhibits incorporation of lysine, phenylalanine, proline and glycine by a cell-free protein-synthesising system from rat liver. Inhibition increases with increasing drug concentration. Phenylalanine incorporation is less depressed than that of other amino acids and is slightly stimulated by lower concentrations of the drug. Liver tRNA, synthetic polynucleotide messengers or ribosomes pre-incubated with HN2 all subsequently show impaired ability to function in the cell-free amino acid incorporating system. Methyl methanesulphonate, 0.9·10 −4 to 1.8·10 −2 M and dimethylnitrosamine, 1·10 −4 to 5·10 −3 M are without significant effect on the ability of the ratliver system to incorporate amino acids. Exposure of uncharged liver tRNA to 2.5·10 −3 M HN2 results in about 30% inhibition of its ability to transfer l-[ 14C]lysine to polylysine in the presence of polyadenylic acid and a liver-ribosomal system. Inactivation of lysine acceptance is of the same order. Preincubation of [ 14C]lysine-charged tRNA with the same concentration of HN2 stimulates the ability of the aminoacyl tRNA both to bind to ribosomes and to transfer lysine to polypeptide in a polyadenylic acid-directed system. It is concluded that inactivation of the acceptor site is the basis for the inhibition of tRNA function by HN2.