THE EFFECT OF DIPHTHERIA TOXIN ON THE METABOLISM OF H E L A CELLS

Recent studies have demonstrated that diphtheria toxin is an enzyme of unusual type. Small amounts of the toxin, added to nicotinamide adenine dinucleotide (NAD)-containing mammalian cell extracts, block peptide-bond formation by catalyzing inactivation of the translocating enzyme, aminoacyltransferase 2 (T2) (1-3). This highly specific reaction involves the splitting of NAD with liberation of free nicotinamide and transfer of the adenosine diphosphate ribosyl moiety (ADPR) to be bound in covalent linkage to T2 (4, 5). Although the reaction is reversible, the equilibrium lies far over on the side of T2 inactivation. Only "free" T2 is inactivated by toxin; ribosome-bound T2 is not attacked (2, 6). The reaction is a highly specific one and no tissue protein other than soluble T2 has been found capable of accepting ADPR from NAD in the presence of toxin. ~ That the same reaction occurs when living ceils in culture are treated with toxin was shown by Gill et al. (5) who found that inactive T2, extracted from intoxicated HeLa cells that were no longer capable of protein synthesis, could be specifically reactivated to full activity by addition of toxin in the presence of excess nicotinamide.Bonventre and Imhoff (8) have reported that when diphtheria toxin is injected into guinea pigs, reduction in the rate of leucine incorporation into protein can be demonstrated in heart and pancreas only. In other organs from intoxicated guinea pigs in which morphological damage is known to occur, they were unable to detect a decreased leucine uptake. More recently, Bowman and Bonventre (9) have examined the rate of 14C-leucine incorporation in extracts of tissue excised from intoxicated guinea pigs and once again concluded that inhibition of protein synthesis is mainly confined to heart muscle. In our laboratory we have failed to observe such organ specificity. In the present

Section: Effect Of Added Toxin On Uc-leucine Incorporation By Normal mentioning

confidence: 99%

Action of Diphtheria Toxin in the Guinea Pig

Baseman

Pappenheimer

Gill

et al. 1970

“…Cells.--When one or more saturating doses of diphtheria toxin are added to a culture of HeLa cells, amino acid incorporation continues at its normal rate for 1.5-2 hr before coming to a complete halt (17)(18)(19). It was of interest to assay the aminoacyl transfer activity in preparations made during this latent period of apparent normal growth from cells treated with excess toxin.…”

Section: Aminoacyl Transferase Activity In Intoxicated Helamentioning

confidence: 99%

Studies on the Mode of Action of Diphtheria Toxin

Gill

Pappenheimer

Brown

et al. 1969

157

In a preceding paper it was shown that low concentrations of diphtheria toxin, in the presence of NAD 1 as an essential cofactor, inhibit the incorporation of amino acids into TCA-precipitable peptides in mammalian cell extracts (1). Collier (2) has demonstrated that this inhibitory action of the toxin is due to the specific inactivation of transferase II, one of the highly labile soluble enzymes involved in transfer of amino acids from aminoacyl=tRNA to the growing peptide chain on the ribosome (3, 4). Goor and Pappenheimer (5) also concluded that transferase II was the site of toxin action and provided evidence that soluble transferase II was the only factor needed for protein synthesis that is lacking in extracts from intoxicated cells. It was further concluded from quantitative studies (6) that toxin and transferase I I interacted stoicbeiometrically in the presence of NAD to form an inactive toxin-transferase II complex.Although the model involving toxin-transferase I I complex formation proposed by Goor et al. (6) appeared to account for most of the quantitative relationships between toxin, NAD, and inhibition of peptide bond formation in cell-free extracts, it failed to explain how a mere 25-50 toxin molecules located in the outer cell membrane (7) could completely block protein synthesis and inactivate all of the soluble transferase II in the living cell within a relatively short period of time. Moreover, the model failed to provide a satisfactory explanation for the striking reactivation of previously intoxicated cell extracts upon addition of nicotinamide (6). Finally, despite repeated efforts, we have been unable to obtain any direct evidence for the postulated toxin-NAD-transferase II complex using either l*~I-labeled toxin or 14C-labeled NAD.

“…HeLa cells are sensitive to diphtheria toxin and were chosen for inclusion in our experiments since they were the tissue culture cells chosen in previous experiments concerning the mode of action of diphtheria to~n (5,6).…”

Section: Materials and Metkodsmentioning

confidence: 99%

Studies on the Mode of Action of Diphtheria Toxin

Bonventre

Imhoff

1966

PLATES 110 ZO 112 (Received for publication 5 August 1966)The factor which confers virulence to specific strains of Corynebacterium dipktheriae is the capacity to synthesize a protein exotoxin in the naso-pharyngeal tissues of infected individuals. The protein toxin has been purified and crystallized and has been characterized extensively both chemically and immunologically (1-3). These studies however have not contributed to an understanding of either the chemical moiety responsible for toxicity or to elucidation of the biochemical mode of action of diphtheria toxin. Presently, hypotheses concerning the biological action of the toxin are based primarily on information derived from tissue culture systems. Indeed, the beautiful experiments of Pappenheimer and Williams (4), who utilized the developing cecropia silkworm as the experimental animal, which pointed to the inhibition of tissue cytochrome systems as the mode of action of the toxin, had to be reevaluated in the light of tissue culture experiments which followed. Strauss and Hendee (5) made the initial significant observation that diphtheria toxin when incubated with HeLa cell cultures caused a virtual cessation of protein synthesis as measured by the incorporation of S~5-methionine into cell proteins. Inhibition of de novo synthesis moreover, occurred several hours before any evidence of the toxin's cytopathic effect became manifest. In view of the fact that the metabolism of HeLa cells (derived from a human neoplasm) is primarily glycolytic in nature, the complete inhibition of protein synthesis by the toxin could not be explained solely by an effect on the aerobic respiratory chain of which the cytochrome system is an integral part. Subsequent experiments by Collier and Pappenheimer (6) who used cell-free protein-synthesizing systems, provided unequivocal evidence that the toxin inhibited de novo protein synthesis mediated by both natural and synthetic messenger RNA. They concluded that cells sensitive *