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.
Aminoacyl transferase II (T2) is required, together with aminoacyl transferase I (Ti), guanosine triphosphate (GTP), and ribosomes, for the transfer of amino acids from aminoacyl tRNA into protein in the cell-free system from rat liver.'-3 Recent investigations 5 indicate that T2 functions in the elongation of polypeptide chains by catalyzing translocation6' 7 of newly lengthened peptidyl tRNA from an unreactive site on the ribosome to a site in which it again can form a peptide bond. The transferase activity of T2, measured in the presence of T1 by the transfer of amino acids from aminoacyl tRNA into protein, is inhibited by diphtheria toxin and nicotinamide adenine dinucleotide (NAD).8-12 Toxin produces this inhibition by catalyzing the transfer of the ADP ribose moiety of NAD to T2.13 The translocase activity of T2, measured in the absence of T1 by a GTP-dependent increase in the number of polypeptide chains which react with puromycin, is also inhibited by toxin and NAD.5In the present report we describe a ribosome-dependent GTPase activity which cochromatographs on Sephadex G 200 with T2. Recently, the same activity, reminiscent of the ribosome-dependent GTPase in the G factor from E. coli,'4-17 has been observed in both rat liver and rabbit reticulocytes by Felicetti and Lipmann.'8 These authors suggest that the activity has a role in protein synthesis, but they point out that its functional significance has not yet been established. We report that the ribosome-dependent GTPase activity of T2, like its activity measured in the transfer and translocation assays, is inhibited by diphtheria toxin in the presence of NAD. In addition, we show that GTP binds to partially purified T2. In the absence of ribosomes, this binding of GTP to macromolecular material is not influenced by diphtheria toxin and NAD, but inhibition of binding is observed when ribosomes are present. We also present data which show that both GTP and ribosomes protect T2 from toxin-catalyzed ADP ribosylation.Inhibition of the ribosome-dependent GTPase by diphtheria toxin, specifically in the presence of NAD, strongly suggests that the hydrolysis of GTP is related to protein synthesis. These data, together with previous results showing similar inhibitions of T2 activity measured in either the transfer or translocation assay, further suggest that the GTPase reaction is involved in translocation of peptidyl tRNA on the ribosome. Materdals and Methods.-Guanosine-5'-triphosphate-,yP'2 (yP32-GTP) was obtained from International Chemical and Nuclear Corporation; guanosine-5'-triphosphate-H' (Hs-GTP), 1.0 c/mmole, from Schwarz BioResearch, Inc.; and C"-L-phenylalanyl tRNA from E. coli (uniformly labeled), 42 myc/mg, from New England Nuclear Corp.; NAD labeled with H3 in the adenosine moiety (H3-NAD) was prepared as described by 1428
It was shown by Collier and Pappenheimer (1) that inhibition, by diphtheria toxin, of amino acid incorporation into polypeptides in cell-free systems isolated from mammalian cells requires the presence of nicotinamide adenine dinucleotide (NAD) as an essential cofaetor. In the absence of NAD, there is no inhibition of amino acid incorporation, even in the presence of high toxin concentrations. More recently, Collier (2) and ourselves (3) have demonstrated that toxin causes inhibition of peptide bond formation (in vitro) by inactivation of a soluble enzyme, transferase II. However, the mechanism by which NAD helps to bring about this inactivation has not been elucidated. The early studies (1) had already suggested that the NAD requirement is highly specific since NAD cannot be replaced by nicotinamide adenine dinucleotide phosphate (NADP).I n the present paper, we are reporting further experiments on the specificity of the N A D requirement for inhibition of amino acid incorporation bydiphtheria toxin in cell-free extracts. A series of N A D analogues have been tested for their ability to replace N A D as a cofactor for inactivation of amino acid transfer by the toxin. The results have been interpreted as indicating that interaction takes place between toxin and certain of the analogues. In preliminary experiments, such interaction has been demonstrated directly, using the method of equilibrium dialysis. Materials and MethodsDiphtheria Toxin.--The purified toxin used contained 2.6 #g protein per Lf unit and 60-70 guinea pig MLD/Lf. The method of preparation was similar to that already described (3).
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