Site in Cell-free Protein Synthesis Sensitive to Diphtheria Toxin

William Johnson, Robert J. Kuchler, Morris Solotorovsky
1968 Journal of Bacteriology  
The effects of diphtheria toxin on cell-free protein synthesis in a bacterial system, and preparations obtained from animals that were sensitive and resistant to toxin were examined. In the presence of nicotinamide adenine dinucleotide (NAD), toxin inhibited the incorporation of amino acids by endogenous and synthetic polynucleotides in both rat liver and guinea pig liver cell-free systems that were exposed to 6 Lf units per ml of toxin. A cell-free system derived from Streptococcus faecalis
more » ... resistant to high concentrations of toxin. Dialyzed toxin-antitoxin floccules that are formed in the presence of NAD and the 105,000 X g supernatant fluid from rat liver contain NAD. Such floccules are also active in protein synthesis in the absence of added transferase I or II. An operational model presents the view that the intoxication complex is formed at the ribosomal level and occurs in two steps. First, the toxin molecule binds to transferase II and alters its stereospecific relationship to transferase I, but it does not result in an inactive complex. Second, the stereospecific alteration in transferase II caused by the binding of diphtheria toxin allows NAD to bridge between transferase I and II, which then results in an inactivated complex. The sensitivity of the cell-free system derived from the normally resistant rat implies that in some cells the cell membrane serves as a permeability barrier to the toxin molecule. The resistance of bacterial cell-free protein synthesizing systems to diphtheria toxin may reflect basic differences between transferase enzymes from bacterial and mammalian sources. The examination of various intact mammalian cells cultured in vitro and the use of cell-free protein synthesizing systems have now provided model systems for study on a molecular level of the mode of action of diphtheria toxin. Strauss and Hendee (21) demonstrated that the first detectable event in intoxicated HeLa cells was the inhibition of protein synthesis. Collier and Pappenheimer (3, 4) confirmed the inhibition of protein synthesis in intact cells and demonstrated a requirement for nicotinamide adenine dinucleotide (NAD) as a cofactor for intoxication in cell-free protein synthesizing systems. Toxin was found to inhibit protein synthesis at a point beyond the formation of aminoacyl-soluble ribonucleic acid (-sRNA), and it was suggested that toxin inhibits the transfer of amino acids from sRNA to the growing polypeptide chain. In mammalian cells, this transfer reaction is dependent on two labile enzymes, transferase I and II (9). Collier (2) has presented strong evidence that toxin, in the presence of NAD, Present address: Biological Laboratories, Fort Detrick, Frederick, Md. 21701. specifically inactivates purified transferase II. The experiments of Goor et al. (11) have confirmed the existence of a toxin-transferase complex. Transferase II was shown to be liberated from toxin-antitoxin floccules that were formed in the presence of NAD and cell extracts. Using cell-free protein synthesizing systems, we have obtained direct evidence that NAD is present in toxin-antitoxin floccules that are formed in the presence of NAD and the 105,-000 X g supernatant fluid from rat liver. Such floccules also have been shown to be active in the polyuridylic acid (poly U)-directed synthesis of polyphenylalanine in the absence of added transferase I or II. Our results suggest a mechanism for inactivation of the transferase molecules, and we propose an operational model for the intoxication reaction. MATERIALS AND METHODS Diphtheria toxin. Crude toxin (Wyeth Laboratories, Marietta, Pa.) containing 100 Lf units and 4,000 minimum lethal doses (MLD) per ml were dialyzed overnight against 0.02 M phosphate buffer (pH 6.9). Diethylaminoethyl (DEAE) cellulose was 1089 Vol. 96, No. 4
doi:10.1128/jb.96.4.1089-1098.1968 fatcat:hsuhvcznwbbyzctswnudet7som