ADP-ribose Transferase Activity Research Articles

Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC(50) = 4.6 +/- 0.4 ng/ml) and crustaceans (Artemia nauplii LD(50) = 10 +/- 2 mug/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-A resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.

Toward the Elucidation of the Catalytic Mechanism of the Mono-ADP-Ribosyltransferase Activity of Pseudomonas aeruginosa Exotoxin A

The catalytic mechanism for the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A was investigated by steady-state and stopped-flow kinetic analyses. The rate constants for binding of the NAD(+) substrate to the enzyme were found to be 4.7 +/- 0.4 microM(-1) s(-1) and 194 +/- 15 s(-1) for k(on) and k(off), respectively. The k(on) and k(off) rate constants for the eEF-2 substrate binding to the enzyme were 320 +/- 39 microM(-1) s(-1) and 131 +/- 22 s(-1), respectively. A potent, competitive inhibitor against the enzyme, 1,8-naphthalimide, bound the enzyme with k(on) and k(off) rates of 82 +/- 9 microM(-1) s(-1) and 51 +/- 6 s(-1), respectively. Furthermore, the binding on and off rates for the reaction products, ADP-ribose and nicotinamide, were too rapid for detection with the stopped-flow technique. Investigation of the pre-steady-state kinetics for the ADP-ribose transferase activity of the toxin-enzyme showed that there is no pre-steady-state complex formed during the catalytic cycle. Binding of NAD+ and smaller compounds representing the various parts of this substrate were investigated by the fluorescence quenching of the intrinsic toxin fluorescence. The binding data revealed a significant structural change in the enzyme upon NAD+ binding that could not be accounted for on the basis of the sum of the structural changes induced by the various NAD+ constituents. Product inhibition studies were conducted with nicotinamide and eEF-2-ADP-ribose, and the results indicate that the reaction involves a random-order ternary complex mechanism. Detailed kinetic analysis revealed that the eEF-2 substrate shows sigmoidal kinetic behavior with the enzyme, and fluorescence resonance energy transfer measurements indicated that wheat germ eEF-2 is oligomeric in solution.

Cholera toxin B subunit activates arachidonic acid metabolism.

Cholera toxin (CT) increases intestinal secretion of water and electrolytes and modulates the mucosal immune response by stimulating cellular synthesis of arachidonic acid (AA) metabolites (e.g., prostaglandin E2), as well as the intracellular second messenger cyclic AMP (cAMP). While much is known about the mechanism of CT stimulation of adenylate cyclase, the toxin's activation of phospholipase A2, which results in increased hydrolysis of AA from membrane phospholipids, is not well understood. To determine whether CT activation of AA metabolism requires CT's known enzymatic activity (i.e., ADP-ribosylation of GSalpha), we used native CT and a mutant CT protein (CT-2*) lacking ADP-ribose transferase activity in combination with S49 wild-type (WT) and S49 cyc- murine Theta (Th)1.2-positive lymphoma cells deficient in GSalpha. The experimental results showed that native CT stimulated the release of [3H[AA from S49 cyc- cells at a level similar to that for S49 WT cells, indicating that GSalpha is not essential for this process. Further, levels of cAMP in the CT-treated cyc- cells remained the same as those in the untreated control cells. The ADP-ribosyltransferase-deficient CT-2* protein, which was incapable of increasing synthesis of cAMP, displayed about the same capacity as CT to evoke the release of [3H]AA metabolites from both S49 WT and cyc- cells. We concluded that stimulation of arachidonate metabolism in S49 murine lymphoma cells by native CT does not require enzymatically functional CT, capable of catalyzing the ADP-ribosylation reaction. These results demonstrated for the first time that stimulation of adenylate cyclase by CT and stimulation of AA metabolism by CT are not necessarily coregulated. In addition, the B subunits purified from native CT and CT-2* both simulated the release of [3H]AA from S49 cyc- cells and murine monocyte/macrophage cells (RAW 264.7), suggesting a receptor-mediated cell activation process of potential importance in enhancing immune responses to vaccine components.

Chromium effects on chondrocytic differentiation in vitro.

A tissue culture study was conducted on the effects of chromium on chondrocytic differentiation. Mesenchymal cells from stage 22-24 chick limb buds were dispersed and cultured as micromasses, where they differentiated into chondrocytes. Addition of chromium(VI) to the cultures indicated that the production of proteoglycans (as detected by Alcian blue staining) was more sensitive to chromium's effects than was cell proliferation. Whereas Alcian blue nodule formation was inhibited by 1 microM Cr(VI), cell proliferation (as detected by cell counts) was not. Chromium (VI) was added to cultures at daily intervals, and these studies indicated that the interval of d 1-2 was the most sensitive period. ADP-ribose transferase activity in these cultures was measured; the pattern of enzyme activity in control cultures was high 1 and 24 h after the start of culture, decreased abruptly between 24 and 48 h, and then decreased more gradually. In the presence of Cr(VI), elevated ADP-ribose transferase levels were maintained throughout the culture period. We suggest that, in presence of 1.0 microM chromium(VI) or higher concentrations, the balance of events favors nucleolytic action rather than repair of damage.

Location and amino acid sequence around the ADP-ribosylation site in the cholera toxin active subunit A 1

Renatured, S-carboxymethylated subunit A 1 of cholera toxin possess the ADP-ribose transferase activity (Lai, et.al., Biochem. Biophys. Res. Commun. 1981, 102 , 1021). In the absence of acceptor self ADP-ribosylation of A 1 subunit was observed. Stoicheometric incorporation of ADP-ribose moiety was achieved in 20 min at room temperature in a 0.1 – 0.2M PO 4(Na) buffer, pH 6.6. On incubation of the complex with polyarginine, 75% of the enzyme-bound ADP-ribose moiety was transferred to the acceptor in 25 min. The ADP-ribosylated A 1 was stable at low pH, and on cleavage with BrCN, the ADP-ribose moiety was found associated with peptide Cn I, the COOH-terminal fragment of A 1 subunit. On further fragmentation with cathepsin D, a dodecapeptide containing ADP-ribose moiety was isolated whose structure was determined as: Asp-Glu-Glu-Leu-His-Arg-Gly-Tyr-Arg ∗-Asp-Arg-Tyr. The Arg ∗ in the peptide was indicated to be the site of ADP-ribosylation.

Intrinsic ADP-Ribose Transferase Activity versus Levels of Mono(ADP-Ribose)Protein Conjugates in Proliferating Ehrlich Ascites Tumor Cells

Transition of proliferating Ehrlich ascites tumor cells (3 days after transplantation) to the non-proliferating status (8--14 days after transplantation) was associated with an increase in total mono (ADP-ribose) protein conjugates. This increase was largely confined to the NH2OH-resistant subfraction. When the amounts of mono-(ADP-ribose) conjugates from 20% trichloroacetic acid precipitates were compared with those from 5% perchloric acid precipitates, no significant differences were seen. This fact excludes histone H1 as a major mono (ADP-ribose) acceptor in vivo in these cells. Transition to the resting state was also associated with a small decrease in NAD levels, and with no significant changes of total ADP-ribose transferase activity. However intrinsic ADP-ribose transferase activity as expressed in permeabilized cells was increased, being correlated with the changes in the level of the NH2OH-resistant mono (ADP-ribose) protein conjugates. This shows that alterations in intrinsic transferase activity may, in general, indicate similar alterations in major subfractions of ADP-ribose conjugates. Intrinsic ADP-ribose transferase activity exhibited an inverse relationship to ornithine decarboxylase activity.

Influence of spermine and histones on a nuclear nucleosidase with ADP-ribose transferase activity in ehrlich ascites cells

Influence of spermine and histones on a nuclear nucleosidase with ADP-ribose transferase activity in ehrlich ascites cells