The quantitative effect of diphtheria toxin on growth and metabolism of mammalian cells in tissue culture was first investigated by Strauss and Hendee (1). They showed that within 2 hr after addition of an excess of toxin, HeLa cells were no longer able to incorporate amino acids into protein, although aerobic glycolysis and oxygen uptake continued at normal rates for several hours thereafter. Later, it was shown by Kato and Pappenheimer (2) and by Strauss (3) that RNA synthesis is maintained and that intoxicated cells continue to take up and concentrate potassium ions from the external medium (2) for a considerable time after growth ceases. Moreover, the ATP and GTP contents of intoxicated HeLa cells remain at their normal levels for many hours after protein synthesis has been arrested (4) and the cells retain their normal morphological appearance. The effect of toxin on protein synthesis in vivo, therefore, seems to be highly specific. Additional confirmation of the specificity of toxin action comes from recent observations by Duncan and Groman (5) who have shown that toxin blocks synthesis of poliomyelitis viral proteins in infected HeLa cells.Collier and Pappenheimer (6) found that incorporation of amino acids into polypeptides by cell-free systems extracted from Helm cells and from rabbit reticulocytes could be inhibited up to 90% or more by low concentrations of toxin, provided that a specific cofactor~ identified as nicotinamide adenine dinucleotide (NAD) was present. Since the toxin was found to be without effect on the activation of amino acids or on the formation of aminoacyl-sRNA, they concluded that inhibition of protein synthesis by toxin takes place at the level of transfer from aminoacyl-sRNA to the growing peptide chain on the ribosomes.Mammalian cells are known to contain at least two soluble and highly labile enzymes that are required for binding of aminoacyl-sRNA to ribosomes and for catalysis of pepfide bond formation (7,8). Gasior and Moldave (8) have termed these

In the preceding paper (1), we reported on the relative ability of various nucleotides related to nicofinamide adenine dinucleotide (NAD) to serve as cofactors for inhibition by diphtheria toxin of protein synthesis in cell-free extracts. Those few analogues which could replace NAD as activators of diphtheria toxin all proved to be nucleotides of demonstrated coenzyme activity. The results suggested that NAD and certain related substances are capable of interaction with the toxin protein. That diphtheria toxin does, in fact, reversibly bind one mole of NAD per mole of toxin was demonstrated by equilibrium dialysis and by gel filtration.In the present paper, we are reporting studies on the quantitative relationships between NAD, diphtheria toxin, and inhibition of peptide bond formation in cell-free extracts from various species. The data have led us to the conclusion that inhibition of protein synthesis, in vitro, results from reversible interaction between three components: toxin, NAD, and transferase II (2, 3). Reduction of NAD is not involved since it has been found that the inactivation of transferase II by toxin in mammalian cell extracts can be prevented and even reversed by relatively low concentrations of nicotinamide. Materials and MethodsReagents and Radioisotopes.--Materials used to determine amino acid incorporation in the cell-free systems were the same as in the preceding papers (1, 3). Nicotinamide, nicotinic acid, and pyridine-3-sulfonic acid were obtained from Nutritional Biochemical Corp., Cleveland,

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.

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