Search

The gastric pathogen Helicobacter pylori activates the NF-κB pathway in human epithelial cells via the recently discovered α-kinase 1 TRAF-interacting protein with forkhead-associated domain (TIFA) axis. We and others showed that this pathway can be triggered by heptose 1,7-bisphosphate (HBP), an LPS intermediate produced in gram-negative bacteria that represents a new pathogen-associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF-κB activation. Instead, we identified ADP-glycero-β-D-manno-heptose (ADP heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram-negative bacteria. ADP heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the β-form, even when the compound was added extracellularly. The data lead us to conclude that ADP heptose not only constitutes the key PAMP responsible for H. pylori-induced NF-κB activation in epithelial cells, but it acts as a general gram-negative bacterial PAMP.-Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling

Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate L-glycero-D-manno-heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6'' position converting ADP-D-glycero-D-manno-heptose into ADP-L-glycero-D-manno-heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP-L-glycero-D-manno-heptose 6-epimerase (AGME). The structure of the enzyme was known but the arrangement of the catalytic site with respect to the substrate is unclear. We now report the structure of AGME bound to a substrate mimic, ADP-beta-D-mannose, which has the same stereochemical configuration as the substrate. The complex identifies the key residues and allows mechanistic insight into this novel enzyme.

New unnatural sugar nucleotides, UDP-Fuc and CDP-Fuc were synthesized from fucose-beta-1-phosphate and nucleotide monophosphates activated as morpholidates. Furthermore, a nucleotide analogue was prepared by phosphorylation of 1-(beta-D-ribofuranosyl)cyanuric acid, itself obtained as a protected derivative by condensation of the persilylated derivative of cyanuric acid with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose in 74% yield. This phosphate activated according to the same procedure was condensed with fucose-beta-1-phosphate, affording a new sugar nucleotide conjugate (NDP-Fuc) which was evaluated together with UDP-Fuc, CDP-Fuc and ADP-Fuc, as fucose donors in alpha-(1-->4/3)-fucosyltransferase (FucT-III) catalyzed reaction. Fucose transfer could be observed with each of the donors and kinetic parameters were determined using a fluorescent acceptor substrate. Efficiency of the four analogues towards FucT-III was in the following order: UDP-Fuc=ADP-Fuc>NDP-Fuc>CDP-Fuc. According to the same strategy ADP-GlcNAc was prepared from AMP-morpholidate and N-acetylglucosamine-alpha-1-phosphate; tested as a glucosaminyl donor towards Neisseria meningitidis N-acetylglucosaminyl transferase (LgtA), ADP-GlcNAc was recognized with 0.1% efficiency as compared with UDP-GlcNAc, the natural donor substrate.

Proteins containing the Nudix box "GX(5)EX(7)REUXEEXGU" (where U is usually Leu, Val, or Ile) are Nudix hydrolases, which catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we report cloning and characterization of a human cDNA encoding a novel nudix hydrolase NUDT5 for the hydrolysis of ADP-sugars. The deduced amino acid sequence of NUDT5 contains 219 amino acids, including a conserved Nudix box sequence. The recombinant NUDT5 was expressed in Escherichia coli and purified to near homogeneity. At the optimal pH of 7, the purified recombinant NUDT5 catalyzed hydrolysis of two major substrates ADP-ribose and ADP-mannose with K(m) values of 32 and 83 microM, respectively; the V(max) for ADP-mannose was about 1.5 times that with ADP-ribose. The murine NUDT5 homolog was also cloned and characterized. mNudT5 has 81% amino acid identity to NUDT5 with catalytic activities similar to NUDT5 under the optimal pH of 9. Both NUDT5 and mNudT5 transcripts were ubiquitously expressed in tissues analyzed with preferential abundance in liver. The genomic structures of both NUDT5 and mNudT5 were determined and located on human chromosome 10 and mouse chromosome 2, respectively. The role of NUDT5 in maintaining levels of free ADP-ribose in cells is discussed.

An efficient one-pot three enzymes strategy for chemoenzymatic synthesis of ADP-d-glycero-β-d-manno-heptose (ADP-d, d-heptose) was reported using chemically synthesized d, d-heptose-7-phosphate and the ADP-d, d-heptose biosynthetic enzymes HldE and GmhB. Moreover, the result of investigating substrate specificity of the kinase action of HldE revealed that HldE had highly restricted substrate specificity towards structurally modified heptose-7-phosphate analogs.

Incubation of ADPribose with yeast phosphoriboisomerase resulted in the formation of an adenylic nucleotide that was identified with ADPribulose by mass spectrometry. Synthesis of [ 32 P]ADPribulose from [ 32 P]NAD + by the combined activities of commercial NAD + glycohydrolase and phosphoriboisomerase allowed us to use it as a labeled internal standard throughout the procedure of purification from trichloroacetic acid extracts of human red blood cells. ADPribulose was purified by means of three sequential reverse phase HPLC separations and its Concentration in human erythrocytes was estimated to be 0.11 ± 0.1 μM. Unsealed erythrocyte ghosts did not transform ADPribulose, which bound to specific membrane proteins with a trichloroacetic and formic acid-resistant binding. Thelabeled proteins were identified as spectrin, bands 3, 4.1, 4.2 and Glyceraldehyde 3-phosphate dehydrogenase on the basis of their relative mobilities on SDS-PAGE.

ADP sugar pyrophosphatase (AspP) is a member of the 'Nudix' (Nucleoside diphosphate linked to some other moiety X) hydrolase family of enzymes that catalyzes the hydrolytic breakdown of ADP-glucose (ADPG) linked to glycogen biosynthesis. In a previous work, we showed that AspP activity is strongly enhanced by both glucose-1,6-bisphosphate and nucleotide-sugars, and by macromolecular crowding. In this work, we show that AspP binds to cell membranes as the bacterial population density increases, c. 30% of the total enzyme remaining membrane associated as glycogen depletes during the stationary phase. This process is not dependent on the stationary transcription factor RpoS, the producer of the bacterial quorum-sensing autoinducer 2 (LuxS), the presence of glycogen granules or glucose availability, but is stimulated by small soluble heat-labile molecule(s) occurring in cell-free spent supernatants of stationary cultures that are acid stabile and base labile. These data further point to AspP as a highly regulated enzyme, and provide a first set of evidences indicating that glycogen metabolism is subjected to regulation by intercellular communication in Escherichia coli.

ADP-l-glycero-d-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the interconversion of ADP-beta-d-glycero-d-manno-heptose (ADP-d,d-Hep) and ADP-beta-l-glycero-d-manno-heptose (ADP-l,d-Hep). The latter compound provides the heptose moiety that is used in lipopolysaccharide biosynthesis by Gram-negative bacteria. Several lines of evidence suggest that the enzyme uses a direct oxidation/reduction mechanism involving a tightly bound NADP+ cofactor. An initial oxidation at C-6'' gives a 6''-keto intermediate, and a subsequent reduction on the opposite face of the carbonyl group generates the epimeric product. The reorientation required for the nonstereoselective reduction could take place within a single active site, or it could involve the release of the intermediate and rebinding in an altered conformation. To distinguish between these possibilities, two isotopically labeled substrates (ADP-d,d-Hep) were prepared that contained 18O and 2H isotopes at C-7'' and C-6'', respectively. A crossover experiment was used to determine whether unlabeled or doubly labeled products were formed upon epimerization of a mixture of the two singly labeled compounds. After an initial epimeric equilibrium was reached, no crossover could be detected, indicating that intermediate release is not intrinsic to the overall mechanism. After extended incubation, however, scrambling of the labels could be detected, indicating that a low background rate of intermediate release does occur. To directly detect the release of the intermediate, the labeled compounds were independently epimerized in the presence of a ketone-trapping reagent, phenylhydrazine. The corresponding phenylhydrazones were identified by mass spectrometry, and the absence of any 2H isotope in the adduct obtained from the deuterated starting compound confirmed that the oxidation had occurred at C-6''.

SummaryThe lipopolysaccharide (LPS)-rich outer membrane of gram-negative bacteria provides a protective barrier that insulates these organisms from the action of numerous antibiotics. Breach of the LPS layer can therefore provide access to the cell interior to otherwise impermeant toxic molecules and can expose vulnerable binding sites for immune system components such as complement. Inhibition of LPS biosynthesis, leading to a truncated LPS molecule, is an alternative strategy for antibacterial drug development in which this vital cellular structure is weakened. A significant challenge for in vitro screens of small molecules for inhibition of LPS biosynthesis is the difficulty in accessing the complex carbohydrate substrates. We have optimized an assay of the enzymes required for LPS heptose biosynthesis that simultaneously surveys five enzyme activities by using commercially available substrates and report its use in a small-molecule screen that identifies an inhibitor of heptose synthesis.

[reaction: see text] A chemoenzymatic synthesis of ADP-D-glycero-beta-D-manno-heptose (ADP-D,D-Hep) is described in which D,D-Hep 7-phosphate is converted to ADP-D,D-Hep by two biosynthetic enzymes. This strategy allows access to the 6''-deuterated analogue, which upon incubation with the epimerase showed complete retention of the isotopic label at the 6''-position. This provides evidence for a direct oxidation mechanism in which the hydride initially transferred to the NADP+ cofactor is subsequently returned to the same carbon in a nonstereospecific manner.

The steps involved in the biosynthesis of the ADP- l - glycero -β- d - manno- heptose (ADP- l -β- d -heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP- d -β- d -heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD ), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP- d -β- d -heptose. Our results demonstrate that the synthesis of ADP- d -β- d -heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional d -β- d -heptose 7-phosphate kinase/ d -β- d -heptose 1-phosphate adenylyltransferase), and GmhB ( d , d -heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP- l -β- d -heptose precursor utilized in the assembly of inner core LPS.

L-Glycero-D-manno-heptopyranose is a characteristic compound of many lipopolysaccharide (LPS) core structures of Gram-negative bacteria. In Escherichia coli two heptosyltransferases, namely WaaC and WaaF, are known to transfer L-glycero-D-manno-heptopyranose to Re-LPS and Rd(2)-LPS, respectively. It had been proposed that both reactions involve ADPL-glycero-D-manno-heptose as a sugar donor; however, the structure of this nucleotide sugar had never been completely elucidated. In the present study, ADPL-glycero-D-manno-heptose was isolated from a heptosyltransferase-deficient E. coli mutant, and its structure was determined by nuclear magnetic resonance spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry as ADPL-glycero-beta-D-manno-heptopyranose. This compound represented the sole constituent of the bacterial extract that was accepted as a sugar donor by heptosyltransferases I and II in vitro.

The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5'-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. K(m) and k(cat) values with ADP-ribose were 60 microM and 5.5 s(-1) respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg(2+) or 100-250 microM Mn(2+) ions; fluoride was inhibitory, with an IC(50) of 20 microM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD(+) and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.

Nuclear protein A24, which is composed of histone H2A and ubiquitin, a nonhistone protein, joined by an isopeptide linkage [ Goldknopf and Busch (1977) Proc . Natl . Acad . Sci . USA 74 , 864–868 ], is found to be ADP-ribosylated in isolated rat liver nuclei.

Initial rate studies at pH 7.6 with three aldehydes, product inhibition patterns with NADH and dead-end inhibition with adenosine diphosphoribose show that the kinetic mechanism of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle cannot be ordered, and support an enzyme-substitution mechanism. Deviations from Michaelis-Menten behaviour are consistent with negative interactions in the binding of NAD+ and instability of the species E(NAD)3 and E(NAD)4. Inhibition with large concentrations of phosphate and arsenate indicates competition for a binding site for glyceraldehyde 3-phosphate, and is not found with glyceraldehyde as substrate.

PERSPECTIVES AND SUMMARY S82 ROLE OF GANGLIOSIDE GMI AS THE CELL SURFACE RECEPTOR FOR CHOLERAGEN ......... ...... . . . ....... . ......... ...... .... 583 Interaction of Gangliosides and Their Oligosaccharides with Choleragen and Its Subunits 584 Activation of Adenylate Cyclase by Choleragen in Intact Cells 585 Activation of Adenylate Cyclase by Choleragen in Cell-Free Systems . 587 REQUIREMENTS FOR DEMONSTRATION OF ADENYLATE CYCLASE ACTIVATION BY CHOLERAGEN IN CELL-FREE SySTEMS ....... . .. 588 NAD !i88 GTP 588 Calcium-Dependent Regulatory Protein 589 ROLE OF NAD AS SUBSTRATE IN REACTIONS CATALYZED BY CHOLERAGEN 589 Mechanism of Action of NAD-Dependent Pseudomonas EXotoxin A and Diphtheria Toxin .... . . . ....... ..... ......... 590 NAD Glycohydrolase and ADP-Ribosyltransferase Activities oj Choleragen 591 Effects ofCholeragen on GTP-Binding Protein and GTPase Activity 592 SIMILARITIES BETWEEN CHOLERAGEN AND ESCHERICHIA COLI HEAT-LABILE ENTEROTOXIN 593 AN ADP-RIBOSYLTRANSFERASE FROM TURKEY ERYTHROCYTES WITH CHOLERAGEN-LIKE ACTIVITy .... . . . . . . . . . . . 595

Measurements of the initial rate of ADP-ribosylation of elongation factor 2 (EF-2) catalyzed by Fragment A from diphtheria toxin support a sequential mechanism and suggest that the reaction proceeds through a central ternary complex involving Fragment A and the substrates, EF-2 and NAD. The Michaelis constants for EF-2 and NAD are 0.15 and 1.4 μM, respectively. As determined by equilibrium gel permeation, EF-2 does not bind Fragment A significantly, alone or in the presence of adenine, ADPribose, nicotinamide or NADH. Based on these and earlier results, we propose an ordered sequential mechanism for the reaction; the sequence of binding of substrates is NAD, followed by EF-2.

A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance (in heterocaryons) and in vitro mixing experiments did not provide evidence that nada mutations alter activators or inhibitors of NAD(P)ase. Thus, the nada gene appears to specify only the structure of N. crassa NAD(P)ase.

Publisher Summary This chapter analyzes poly(ADP-ribose) and ADP-ribosylation of proteins. Poly(ADP-ribose) and the ADP-ribosylation of proteins constitute a novel type of covalent modification of proteins. They are ubiquitously distributed in nature and are implicated in the regulation of cell proliferation, protein synthesis, and DNA as well as RNA metabolism. This type of post-translational modification is unique because NAD, nicotinamide adenine dinucleotide, whose primary function is an electron carrier in biological oxidation, invariably provides the ADP-ribosyl moiety, which is transferred on to a protein molecule. The ADP-ribosyl unit thus, covalently attached to a protein acceptor is present as either a monomer or a polymer as in the case of the nuclear system. This chapter also summarizes developments in this field of research and some experimental results. It reviews briefly, mono ADP-ribosylation of proteins, in which only a single ADP-ribosyl moiety is transferred to a protein acceptor. It also covers a more complex reaction, poly(ADP-ribose) in nuclei, in which the ADP-ribosyl units are polymerized.

In the presence of ATP and a cytosolic factor, cholera toxin fragment A1 catalyzes the transfer of ADP-ribose from NAD to a number of soluble and membrane-bound proteins of the pigeon erythrocyte. Evidence is presented that suggests that the most readily modified membrane protein (Mr 42,000) is the adenylate cyclase-associated GTP-binding protein. Its modification by toxin is stimulated by guanine nucleotides. Adenylate cyclase activity increases in parallel with the addition of ADP-ribose to this protein and decreases in parallel with the subsequent reversal of ADP-ribosylation by toxin and nicotinamide. The protein is only accessible to toxin A subunits if the erythrocytes are lysed. When adenylate cyclase activity reaches a maximum, the number of ADP-ribose residues bound to this protein (about 1500 per cell) is similar to the reported number of beta-adrenergic receptors.

From a Shigella sonnei R mutant which incorporates into its cell wall lipopolysaccharide D-glycero-D-mannoheptose and contains no L-glycero-D-mannoheptose, a nucleotide-linked sugar was isolated and identified as adenosine 5'-diphosphate-D-glycero-d-mannoheptose by chemical and chromatographic analysis. This intermediary compound is assumed to play a role in heptose biosynthesis of Enterobacteria.

ADP-ribosyltransferase activity associated with chromatin is two- to tenfold higher in simian virus 40 (SV40)-transformed cells than in untransformed cells. When confluent transformed cells were subcultured, their specific enzyme activity first decreased two- to fourfold and the rapidly increased during the logarithmic phase of growth. This increase ceased or slowed down when the cells entered the stationary phase. In contrast, the activity in the untransformed cells remained low throughout the growth cycle. In SV40tsA-transformed cells (ts = temperature sensitive), this density-dependent increase in the enzyme activity was observed when the cells were cultivated at the permissive temperature, whereas the activity remained low at the restrictive temperature. The enzyme activity did not increase during induction of cellular DNA synthesis in quiescent cells either by addition of fresh medium or by infection with SV40. The chromatin-associated enzyme activity extracted with 1 m NaCl was eluted together with almost all the DNA-binding proteins from a phosphocellulose column with 0.6 m NaCl. The enzyme activity in this fraction from transformed cells, measured with or without added DNA and histones, was higher than that in a similar fraction from untransformed cells, reflecting the difference in the original activities present in the nuclei of these cells. The chain lengths of poly(ADP-ribose) formed by chromatin from SV40-transformed and untransformed cells were not significantly different. These results suggest that the number of initiation sites for ADP-ribosylation is increased in the chromatin of SV40-transformed cells compared to that of untransformed cells.

We tested various methods of assaying the ADP-ribosyltransferase activity of cholera toxin using artificial acceptors of the ADP-ribosyl group. Any of several proteins or poly(L-arginine) could be used with [adenine-14C]NAD+ as ADP-ribosyl donor, but this method was not ideal because of the heterogeneity of potential acceptor groups and the necessity of using costly labeled NAD+. We, therefore, developed an alternative assay using a synthetic low molecular weight acceptor, 125I-N-guanyltyramine (125I-GT). 125I-GT was specifically ADP-ribosylated by thiol-treated cholera toxin or its A1 peptide in the presence of beta-NAD. ADP-ribosyl-125I-GT was quantified after separation from unreacted 125I-GT by batch absorption of the latter to cation exchange resins. Analysis of the kinetics of ADP-ribosylation of 125I-GT indicated that the reaction proceeds by a sequential rather than a ping-pong mechanism. The Km values for NAD+ and 125I-GT were 3.6 mM and 44 microM, respectively. L-Arginine was a competitive inhibitor of 125I-GT (KI = 75 mM), but was at least 1000-fold less active than 125I-GT as an ADP-ribose acceptor.

Penicillium charlesii extracts contain UDP-galactose:NAD+ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH4 followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D- glycero -D- glycero -3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.

Purified rat liver nuclei were incubated in vitro with [3H]NAD. Altered patterns of ADP-ribosylation of nuclear proteins occurred with 1 mM spermidine or spermine with the latter polyamine causing the greater change. Spermine treated nuclei showed a two-fold increase in ADP-ribose incorporation into H1 histones and a decrease in the other histones. Likewise, the incorporation into the more acidic non-histone nuclear proteins was greater with spermine than spermidine. These results suggest that polyamines may exert a regulatory function by altering the pattern of ADP-ribosylation of both histone and non-histone nuclear proteins.

ADP-Ribose is nearly quantitatively split to 5'-AMP by treatment with alkali at elevated temperatures. This unique behaviour, which is not shown by ADP and other adenine derivatives, was used as the basis of an optical test for the selective determination of ADPR in the presence of other adenine compounds including RNA. Poly(ADPR) could also be quantified when the polymer was degraded by poly(ADPR) glycohydrolase prior to alkaline treatment. When combined with the determination of the terminal AMP residues released by phosphodiesterase I treatment, the chain length of the polymer could be calculated. Application of the method to the quantitation of protein-bound mono(ADPR) residues in Ehrlich ascites tumor cells under different growth conditions is described.

A specific and sensitive radioimmunoassay for ADP-ribose has been developed on the basis of the selective conversion of ADP-ribose to 5′-AMP by alkaline treatment. Antibodies highly specific against 5′-AMP allowed quantification of ADP-ribose converted to 5′-AMP in the range of 1–40 pmol, and in the presence of large quantities of nucleic acids or 3′-AMP. Poly(ADP-ribose) could also be determined when degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. Determination of the chain length of purified polymer was possible by a parallel determination of ADP-ribose residues after glycohydrolase treatment and of 5′-AMP from the nonreducing end obtained by phosphodiesterase catalyzed hydrolysis. The high specificities of the alkaline conversion of ADP-ribose to 5′-AMP and of the radioimmunoassay for 5′-AMP allowed quantification of protein-bound ADP-ribose residues in crude tissue extracts as verified by comparison with chromatographically purified samples.

The exotoxin of Pseudomonas aeruginosa is a proenzyme possessing latent adenosine diphosphate ribose-transferase activity. Conversion to the active form can be effected by simultaneous treatment with a protein denaturant and a chemical able to split disulfide bonds. Activation results from a conformational change that exposes the previously buried active site. Proteolysis is not required.

Aldono-l,S-lactones are efficient competitive inhibitors with high specificity for glycosidases that catalyze the hydrolysis of glycosides derived from the same aldoses [ 11. The inhibition has been attributed to a conformational (half-chair) and electronic (sp2 hybridization and electron delocalization) similarity between the lactone and the hypothetical glycosyl-ion which is involved as an intermediate in substrate transformation [2]. A rationale was provided by application of absolute reaction rates theory to enzymatic catalysis. It predicts that an enzyme develops an enhanced affinity for the transition state of a catalyzed reaction, and therefore for ‘transition state analogues’, comparatively to its substrates [3]. A classical example for glycosidases is that of lysozyme where the glycosyl group at which cleavage occurs must be distorted, according to X-ray analysis, into a half-chair conformation in order to fit into the active site. This substrate distortion towards the structure of the transition state is believed to be a major factor responsible for the mode of action of that enzyme [4]. A 1,5-lactone derived of chitotetraose, an analogue for the proposed transition-state, was found to a potent ligand for lysozyme [ 51. NAD-glycohydrolases (EC 3.2.2.6) catalyze the hydrolysis of NAD at the nicotinamide-ribose linkage. The kinetic mechanism of calf spleen NAD glycohydrolase was found to be consistent with an Ordered Uni Bi, the formation of an ADP-ribosyl intermediary complex (E-ADP-rib) being rate determining [6]. The reactivity of E-ADP-rib versus nucleophiles was in favor of an oxocarbonium ion intermediate in the NAD transformation [6,7] . In

Glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate:nicotinamide adenine dinucleotide oxidoreductase (phosphorylating), EC 1.2.1.12), isolated from rat skeletal muscle undergoes a rapid inactivation upon incubation at 25 °C in the presence of adenine nucleotides. The reaction can be described as a reversible tetramerdimer equilibrium, only the tetrameric form of the enzyme being active in the presence of nucleotides. The standard free energy changes upon dissociation at 25 °C in 0.1 m phosphate buffer pH 7.5 in the presence of saturating concentrations of ATP, ADP, AMP, and ADP-ribose were found to be 6.69, 6.93, 8.31, and 10.5 kcal/mol, respectively. Nucleotide-dependent inactivation does not bring about any alteration of the reactivity of SH groups of the enzyme towards 5,5′-dithiobis(2-nitrobenzoic acid). This is not the case, however, when the enzyme undergoes NaCl-induced cold inactivation, which is accompanied by an increased accessibility of SH groups. ADP and ATP protect the enzyme against cold inactivation in the presence of NaCl and decrease the enhanced reactivity of SH groups. Adenine nucleotide-induced inactivation is prevented in the presence of NAD. The protective effect is noncooperative, the extent of inactivation being dependent upon the amount of active centers free of bound coenzyme. Addition of excess NAD to the inactivated enzyme results in a complete regain of activity. A comparative study made on the rate of reforming enzyme NAD complex (followed spectrophotometrically) and the regain of activity has demonstrated that the former process is markedly more rapid than the latter. The reactivation was observed to follow second-order kinetics, which suggests that the reassociation of the inactive NAD-liganded dimers is the rate-limiting step. The data are consistent with the existence of different conformational transitions responsible for the restoration of the intersubunit contact area, catalytic activity, and thermal stability of the enzyme molecule, respectively.

1. ADP-ribose is found in rat liver nuclei covalently bound to histone F1, to a non-histone protein, and to a small peptide. 2. A single unit of ADP-ribose, covalently bound to phosphoserine, was isolated from an enzymic hydrolysate of histone F1. ADP-ribose-bearing peptides were isolated from a tryptic digest of the histone. 3. It is proposed that the 1′-hydroxyl group of ADP-ribose is linked to the phosphate group of phosphoserine in histone F1. 4. The incorporation of 32P into ADP-ribose on histone F1 a parallels the DNA content through the cell cycle. An increased incorporation of the nucleotide into the other derivatives is observed during S phase. 5. It is suggested that the ADP-ribose derivative of histone F1 has a role in maintaining the G0 state and that one or both of the other derivatives is concerned with control of DNA synthesis.

A nontoxic peptide (molecular weight, 26,000), which is active in catalyzing the adenosine diphosphate (ADP)-ribosylation of elongation factor 2, has been isolated from the culture supernatant of Pseudomonas aeruginosa strain 103 in stationary phase. Like fragment A from diphtheria toxin, the active peptide catalyzed the hydrolysis of nicotinamide adenine dinucleotide as well as the ADP-ribosylation of elongation factor 2 and showed similarities to fragment A in specific activity, kinetic constants, pH optimum, and ionic sensitivity. These results provide strong evidence for a high degree of homology in the structures of their active sites. That the peptide is not identical to fragment A is shown by the fact that it was not neutralized by fragment A-specific antiserum and was different in amino acid composition and pH and thermal labilities. Although definitive evidence is lacking, there are data suggesting that this peptide is a proteolytic fragment from the ADP-ribosylating toxin (exotoxin A; molecular weight, 66,000) produced by the same strain of P. aeruginosa.

After mutagenesis with nitrosoguanidine and selection by immuno-halo techniques, an avirulent mutant, designated Texas Star-SR, which produces no detectable A (active; ADP-ribosylating) region of the cholera enterotoxin (choleragen) but produces the B region (choleragenoid) in amounts similar to the hypertoxinogenic wild-type parent Vibrio cholerae (biotype E1 Tor serotype Ogawa), has been isolated. The mutant retains the colonizing ability, motility, prototrophy, and serologic characteristics of the parent. In relevant intestinal experimental models, it has been shown to be avirulent and to induce protection against challenge with virulent cholera vibrios. The mutant appears to be suitable for further evaluation in volunteers as a candidate living enteric vaccine against cholera and related enterotoxic enteropathies.

Escherichia coli ADP-sugar pyrophosphatase (AspP) is a “Nudix” hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to glycogen biosynthesis. Moderate increases of AspP activity in the cell are accompanied by significant reductions of the glycogen content. In vitro analyses showed that AspP activity is strongly enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars, providing a first set of indicative evidences that AspP is a highly regulated enzyme. To our knowledge, AspP is the sole bacterial enzyme described to date which is activated by both G1,6P2 and nucleotide-sugars.

ADP-ribosylation of nuclear proteins in vitro was first shown by Hayaishi’s group [I] , subsequent work from Dietrich et al. [2] placing special emphasis on the existence of mono(ADPR) residues rather than poly(ADPR) in the protein conjugates. However, secondary degradation of oligoor poly (ADPR) residues to the monomer has not been ruled out in these experiments. In vitro experiments using short-term incubation of nuclei followed by immediate inactivation of enzymes revealed considerable differences in tissues with widely differing proliferation rates, in the levels of monoADP-ribosylated nuclear proteins susceptible to NH2 OH [3] . We were interested in finding out whether this in vitro correlation reflected the in vivo situation. Since no method was available for the quantitation of mono(ADPR) residues we have developed an isotope dilution procedure for measuring NH2 OH-sensitive, proteinbound mono(ADPR), and in this communication report on the endogenous levels of such ADPR residues in different tissues. The data show rather low concentrations of mono(ADPR) residues in vivo, reaching only l-5% of the level of its precursor NAD. Proliferating tissues have significantly lower monoADP-ribosylated proteins than adult rat liver.

Choleragen selectively incorporates 3H from [3H]NAD labeled on the adenosine moiety and not 14C from [14C]NAD labeled on the nicotinamide moiety. This reaction does not require protein in addition to choleragen. Incorporation of isotope does not proceed at 4 degrees, requires dithiothreitol, is stable after extensive washing with cold trichloroacetic acid, and is decreased 80% by boiling in trichloroacetic acid. Studies with the A and B subunits of choleragen show that the A subunit catalyzes ADP-ribosylation and serves as an acceptor protein. The B subunit does not show catalytic or acceptor activity. We conclude that choleragen and its A subunit catalyze the hydrolysis of NAD and the enzymatic transfer of ADP-ribose to the A subunit.

1. 1) Membrane preparations from human red cells are able to lower in vitro the isoelectric pH of the enzyme glucose-6-phosphate dehydrogenase in the presence of the coenzyme NADP + . This action can be obtained through a dialysis bag, and, therefore, involves a dialysable product of NADP + degradation. 2. 2) This product could be identified as phosphoadenosine diphosphoribose by chromatographic isolation. The split product of NADP + was able to provoke anodisation of the enzyme, as was also an authentic sample of phospho-ADP ribose. ADP ribose and 2′–5′ adenosine diphosphate were inactive 3. 3) Experiments performed with a fluorescent analogue of NADP + enabled us to deduce that phospho ADP ribose binds strongly to glucose 6 phosphate dehydrogenase. We conclude that a reaction of phospho ADP ribosylation of glucose 6 phosphate dehydrogenase takes place under in vitro conditions and may play a role in the in vivo appearance of hyperanodic bands of the enzyme in cells.

Treatment of pigeon erythrocyte membranes with cholera toxin and NAD + enhanced the GTP stimulation and suppressed the F - activation of the adenylate cylase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. In the presence of NAD + labeled with 32 P in the AMP moiety the toxin catalyzed the covalent incorporation of radioactivity into membrane proteins with molecular weights ( M r s) of 200,000, 86,000, and 42,000. Extraction of toxin-treated membranes with Lubrol PX followed by affinity chromatography on a GTP-Sepharose column resulted in a 200-fold purification of the 42,000- M r labeled protein and in its complete separation from the other labeled proteins. The fraction containing the purified GTP-binding component from toxin-treated membranes conferred an enhanced GTP-stimulated activity on adenylate cyclase solubilized from nontreated membranes. Likewise, the addition of GTP-binding fraction from nontreated membranes to an enzyme solubilized from toxin-treated membranes restored F - stimulation of the adenylate cyclase. The toxin-induced modification of adenylate cyclase and the incorporation of radioactivity into the 42,000- M r protein were partially reversed upon incubation with toxin and nicotinamide at pH 6.1. The results indicate that cholera toxin affects the adenylate cyclase system by catalyzing an ADP-ribosylation of the 42,000- M r component bearing the guanyl nucleotide regulatory site.

Upon incubation of lysed pigeon erythrocytes with NAD, adenosine diphosphate-ribose (ADP-ribose) is incorporated into nuclear poly ADP-ribose and into an unidentified acid-insoluble product of the cytosol. The properties of these incorporations have been examined and a method developed for reducing their amount whilst retaining the sensitivity of the lysate to cholera toxin. This method has allowed the detection and description of a set of cholera toxin-specific ADP-ribose transfers to membrane-bound and soluble proteins under conditions that lead to adenylate cyclase activation.

We tested various methods of assaying the ADP-ribosyltransferase activity of cholera toxin using artificial acceptors of the ADP-ribosyl group. Any of several proteins or poly(L-arginine) could be used with [adenine-14C]NAD+ as ADP-ribosyl donor, but this method was not ideal because of the heterogeneity of potential acceptor groups and the necessity of using costly labeled NAD+. We, therefore, developed an alternative assay using a synthetic low molecular weight acceptor, 125I-N-guanyltyramine (125I-GT). 125I-GT was specifically ADP-ribosylated by thiol-treated cholera toxin or its A1 peptide in the presence of beta-NAD. ADP-ribosyl-125I-GT was quantified after separation from unreacted 125I-GT by batch absorption of the latter to cation exchange resins. Analysis of the kinetics of ADP-ribosylation of 125I-GT indicated that the reaction proceeds by a sequential rather than a ping-pong mechanism. The Km values for NAD+ and 125I-GT were 3.6 mM and 44 microM, respectively. L-Arginine was a competitive inhibitor of 125I-GT (KI = 75 mM), but was at least 1000-fold less active than 125I-GT as an ADP-ribose acceptor.

The 13C NMR spectra of poly(adenosine diphosphate ribose), ribosyl adenosine 5', 5''-bis(phosphate) and related compounds were analyzed. The structure of the ribose-ribose linkage was determined as alpha-(1'' leads to 2')ribofuranosyl ribofuranoside, from the 13C chemical shifts of methyl-alpha- and methyl-beta-D-ribofuranosides, and from the downfield displacements of 13C NMR signals by glycosidic bond formation.

Pseudomonas aeruginosa exoenzyme S is an adenosine diphosphate ribosyltransferase distinct from Pseudomonas toxin A. Exoenzyme S catalyzes the transfer of radioactivity from all portions of radiolabeled NAD+ except nicotinamide. Digestion of the radiolabeled product(s) formed in the presence of [adenine-14C]NAD+ and exoenzyme S with snake venom phosphodiesterase yields only AMP, suggesting that ADP-ribose is present as monomers and not as poly(ADP-ribose). Exoenzyme S does not catalyze the transfer of ADP-ribose from NAD+ to elongation factor 2, as do toxin A and diphtheria toxin, but to one or more other proteins present in crude extracts of wheat germ or rabbit reticulocytes and in partially purified preparations of elongation factor I. The ADP-ribosyltransferase activity of exoenzyme S is distinct from toxin A by several tests: it is not neutralized by toxin A antibody, it is destroyed rather than potentiated by pretreatment with urea, and it is more heat stable. These latter observations and the substrate specificity suggest that exoenzyme S is different from any previously described prokaryotic ADP-ribosyltransferase.

Covalently bound adducts of ply(L-lysine), bovine serum albumin, lysine rich histone (f1) and deoxyribonucleotidase I (DNase, EC 3.1.4.5) with adenosine diphosphoribose and ribose-5-phosphate were prepared at pH 7.4 and 9.5. Macromolecular adducts of bovine serum albumin and histone (f1) were isolated by gel filtration and electrophoresis. Reduction of products by NaBH4 did not dissociate the ribose-5-phosphate moiety from macromolecules. Specific introduction of 3H into the adducts also indicated Schiff base formation. The reaction of ribose-5-phosphate with epsilon-amino groups of histone (f1) approached 70-90% saturation. Spermine and spermidine also react with adenosine diphosphoribose and ribose-5-phosphate to form 1:1 Schiff bases. It is proposed that high turnover of cellular NAD+ is the source of aldehydic metabolites which may regulate macromolecular metabolism by covalent modification of nuclear proteins, whereas polyamines serve as modulators of this control cycle.

The binding of NAD+, NADH and adenosine diphosphoribose (Ado-PP-Rib) to a stable, highly active and nucleotide-free preparation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate : NAD+ oxidoreductase(phosphorylating), EC 1.2.1.12) has been studied. All three nucleotides quench the protein fluorescence to the same extent when they bind to the enzyme, and this property has been used to measure the dissociation constants for the two high-affinity binding sites for the nucleotides. The results indicate negative interactions between, or non-identity of, these two binding sites, to which NAD+ and NADH bind with similar affinity. The binding of NAD+ to the enzyme has been studied by spectrophotometric titrations of 360 nm. It appears that the binding of NAD+ to each of the four subunits of the enzyme contributes equally to the intensity of this ‘Racker’ band. The dissociation constants associated with the binding of the third and fourth molecules of NAD+ estimated from such titratons confirm some previous estimates. The binding of NADH to the enzyme causes a decrease of intensity of the absorbance of the coenzyme at 340 nm, and the dissociation constants for binding of the third and fourth molecules of NADH have been estimated from spectrophotometric titrations. They are the same as those for NAD+. Judging by the apparent dissociation constants, negative interactions on binding the third molecule of NAD+ or NADH are more marked than those associated with the binding of the second and fourth molecules, suggesting that a major conformational change occurs at half-saturation of the tetramer with coenzyme.

The fluorescence of the natural coenzyme, NADH, is used to monitor the environment of the nicotinamide moiety at the active centre of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). Changes of the fluorescence quantum yield and polarization of a small amount of NADH, totally bound by an excess of enzyme, show that at half-saturation of the oligomer with NAD a conformational change is induced which affects the active centre regions of the remaining subunits. This conformational transition is not effected by adenosine diphosphoribose, suggesting that the binding of the nicotinamide moiety of NAD to two subunits is essential for the change of tertiary structure of the remaining subunits that causes the observed changes of the fluorescence properties of the ADH "tracer probe". It is suggested that this conformational transition of the oligomer is responsible for the major decrease of affinity for NAD which occurs at half-saturation, and possibly for the activation by NAD+ of the reductive dephosphorylation reaction catalysed by the enzyme. It is also suggested, by analogy with haemoglobin, that the molecular basis of the negative cooperativity may be the creation of additional intersubunit bonds during the binding of the first two NAD molecules to the tetramer, and a change from a "relaxed" quaternary structure to a "tense" structure at half-saturation.