Response of cultured mammalian cells to diphtheria toxin. 3. Inhibition of protein synthesis studied at the subcellular level.

Diphtheria toxin inhibited protein synthesis in intact KB cells. The action of the toxin upon the cell did not result in disaggregation of polyribosomes, or in impairment of their ability to function in protein synthesis. A reduction in single ribosomes and a concomitant increase in polyribosomes did result from the action of toxin. Nascent peptides were not cleaved from polyribosomes by the action of toxin, but treatment of fully intoxicated cells with puromycin resulted in cleavage of these peptides, and caused accelerated polyribosome breakdown. Our data indicated that the toxin must enter the cell to exert its effect. The component or components sensitive to toxin were localized in the 100,000 x g supernatant fraction of cytoplasmic extracts. When extracts from intoxicated cells were treated with nicotinamide, a significant proportion of their capacity to synthesize protein was restored. The specificity of this reaction suggested that nicotinamide adenine dinucleotide is involved in the action of toxin in the intact cell, and that one component inactivated by toxin is soluble transferase II.