Intracellular distribution of 8-14C-puromycin aminonucleoside in ultraviolet irradiated Escherichia coli.

The uptake of 8-14C-puromycin aminonucleoside (8-14C-PAN) was studied in ultraviolet (UV) irradiated strains of E. coli B/r hcr+ and hcr-. The cells took up only 0.1-0.3% of the 8-14C-PAN present in the medium when grown in minimal (M9) containing 8-14C-PAN. When ethylenedinitrilotetra-acetic acid (EDTA) treated E. coli cells are placed in a medium containing 8-14C-PAN, the total concentration of 8-14C-PAN in the cells reaches 43-54% of the medium within 30 min of incubation. Almost all 8-14C-PAN can be dialyzed from cells exposed in the absence of an energy source, but cells metabolizing in M9 medium during exposure can retain up to 30% of their internal concentration. Bacteria grown in the presence of 8-14C-PAN, accumulated the radioactive material intracellularly in three forms, namely, unbound, reversibly bound (dialyzable) and irreversibly bound to the protein (nondialyzable). Approx. 70-77% of the irreversibly bound radioactive material linked with the protein fraction was released by treatment with a protease. Addition of PAN into the post-irradiation medium of EDTA-treated hcr+ cells, increased UV induced mutation rates. Antimutagenic purine ribosides decreased the final level of 8-14C-PAN accumulated by the cells. Decreases in 8-14C-PAN uptake in the presence of antimutagens correspond to reductions in the rate of mutation to streptomycin resistance induced by UV light. Therefore, protein bound PAN appears to be the relevant component in the enhancement of UV induced mutation by this drug.