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At least 19 recordsLinked to original sources
PubMed1979

Enzymic activity of cholera toxin. I. New method of assay and the mechanism of ADP-ribosyl transfer.

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.

Adenosine Diphosphate Sugars↗
PubMed1979

Effect of thymidine on poly(ADP-ribosyl)ation in vivo.

The addition of thymidine as well as nicotinamide to isolated nuclei resulted in a strong inhibition of poly(ADP-ribosyl)ation, whereas that of hydroxyurea and amethopterin has essentially no effect. The nuclei isolated from the cells immediately after release from thymidine synchronization exhibited a significantly increased activity of poly(ADP-ribosyl)ation. Thereafter, the fluctuation pattern of the activity of poly(ADP-ribosyl)ation in isolated nuclei during the cell cycle was essentially the same as in the case of hydroxyurea synchronization. The activity of poly(ADP-ribosyl)ation in isolated nuclei after treatment with thymidine in vivo increased with the treatment time. The time-dependent increase was also evident in the case of nicotinamide treatment. Little increase in the activity was observed in hydroxyurea and amethopterin treatment. When poly(ADP-ribose) polymerase was extracted from the nuclei isolated from the cells which were pretreated with each of the four compounds, there was no significant difference in the amount among these compounds. The reason for the increase in the poly(ADP-ribosyl)ation in vitro by the in vivo treatment with thymidine is discussed.

Cell Nucleus↗
PubMed1979

NAD-dependent ADP-ribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (labile toxin, LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the ADP-ribosylation of arginine (Moss, J., and Richardson, S.H. (1978) J. Clin. Invest. 62, 281-285). Analysis of the product of the ADP-ribosylation of arginine by nuclear magnetic resonance spectroscopy indicated that the reaction was stereospecific and resulted in the formation of alpha-ADP-ribosyl-L-arginine. This reaction product rapidly anomerized to yield a mixture of the alpha and beta forms. In the presence of [adenine-U-14C]NAD, E. coli enterotoxin catalyzed the transfer of the radiolabel to proteins; the ADP-ribosylation of proteins was inhibited by arginine methyl ester, an alternative substrate. Digestion of the 14C-protein with snake venom phosphodiesterase released predominantly 5'-AMP. No product was obtained with a mobility similar to that of 2'-(5''-phosphoribosyl)-5'-AMP. This result is consistent with the covalent attachment by the enterotoxin of ADP-ribose rather than poly(ADP-ribose) to protein. Thus, LT is catalytically equivalent to choleragen, an enterotoxin of Vibrio cholerae, and activates adenylate cyclase through a similar stereospecific ADP-ribosylation reaction.

Adenosine Diphosphate Sugars↗
PubMed1976

Poly ADP-ribosylation of DNA-dependent RNA polymerase I from quail oviduct. Dependence on progesterone stimulation.

Progesterone causes in goblet cells of oviducts of estrogen hormone-stimulated immature quails selectively gene activation without affecting DNA synthesis. This biological model has been used to study the influence of poly ADP-ribosylation during the processes of DNA transcription. Administration of progesterone in vivo causes an increase of the activity of RNA polymerase I and II in isolated nuclei. This increase is accompanied by a marked decrease of the specific activity of poly (ADP-Rib) polymerase. After in vitro ADP-ribosylation of nuclear proteins the template capacity of chromatin for ""exogenous'' RNA synthesis (with E. coli DNA-dependent RNA polymerases) as well as for ""endogenous'' RNA synthesis with DNA dependent RNA polymerases II is not affected, whereas the data presented seem to indicate that the capacity for RNA synthesis mediated by ""endogenous'' DNA-dependent RNA polymerase I might be inhibited after ADP-ribosylation. Evidence is presented to show that a considerable amount of poly (ADP-Rib), synthesized by poly (ADP-Rib) polymerase in isolated nuclei, is linked with RNA polymerase I. The rate of synthesis of poly (ADP-Rib) is dependent on the incubation temperature (optimum at 25 degrees C) and it can be inhibited by the specific inhibitors of poly (ADP-Rib) polymerase nicotineamide, thymidine and formycin B. Poly (ADP-Rib) is probably associated with RNA polymerase I through a covalent linkage. ADP-ribosylated RNA polymerase I has been purified 550 fold with respect to the nuclear extract corresponding to a 4,000 fold purification from the whole cell homogenate. The ratio between poly (ADP-Rib), formed during preincubation of nuclei with NAD, and RNA polymerase I remains almost constant during the purification procedures. The extent of ADP-ribosylation of RNA polymerase I decreases during gene expression. Thus we conclude that poly ADP-ribosylation of this enzyme is one of the regulatory mechanisms by which specificity of DNA transcription is achieved.

Animals↗
PubMed1975

ADP-ribosylation of DNA-dependent RNA polymerase of Escherichia coli by an NAD+: protein ADP-ribosyltransferase from bacteriophage T4.

A protein from bacteriophage T4 responsible for the alteration of host DNA-dependent RNA polymerase and absent in T4 alt- phage was purified from T4 phage and enriched from T4-infected cells. It is injected during infection together with the known internal proteins. It has a molecular weight of about 70000 and catalyses the release of nicotinamide and the transfer of the ADP-ribosyl moiety from NAD+ to arginyl residues of various proteins including itself. RNA polymerase from Escherichia coli accepts ADP-ribosyl residues in all four subunits; the alpha subunit reacts with very high specificity. Only half of the alpha subunits are labelled, 45% with one, 5% with two residues. The main product shows the same electrophoretic mobility as alpha subunits altered or modified in vivo. The alpha subunit in modified RNA polymerase is no acceptor.

Bacterial Proteins↗
PubMed2026

NAD+ Metabolism Licenses Zygotic Genome Activation via PARP7-Mediated ADP-Ribosylation of UHRF1 in Mouse Early Embryos.

Zygotic genome activation (ZGA) is a critical developmental milestone whose metabolic regulation remains unclear. This study identifies a pivotal role for Nicotinamide adenine dinucleotide (NAD+) metabolism in regulating ZGA through poly(ADP‑ribose) polymerase 7(PARP7)-mediated ADP-ribosylation. Using ultra-low input embryo metabolomics, we profiled metabolism from zygote to blastocyst, revealing a significant NAD+ decline at the 2-cell stage. This shift coincided with specific upregulation of the mono-ADP-ribosyltransferase PARP7, confirmed by transcriptomics, quantitative RT-PCR, western blot, and immunofluorescence. Genetic knockdown via trim-away technology or pharmacological inhibition with RBN-2397 caused developmental delay/arrest at the 2-cell stage, impaired blastocyst formation, and defective ZGA. Mechanistically, PARP7 deficiency reduced chromatin accessibility (ATAC-seq), diminished H3K4ac and H3K27ac marks, and impaired RNA polymerase II transcription. Integrated proteomics and ADP-ribosylome analysis of late 2-cell embryos identified UHRF1 as a key PARP7 target, mono-ADP-ribosylated at lysines K30 and K31. This modification stabilized UHRF1 protein (cycloheximide chase), and UHRF1 overexpression partially rescued the transcriptional defects associated with ZGA from PARP7 inhibition. Our findings establish a metabolic-epigenetic axis wherein NAD+ metabolism, via PARP7-mediated ADP-ribosylation of UHRF1, regulates chromatin remodeling and transcriptional activation during ZGA, offering fundamental insights into early development.

Animals↗
PubMed1978

Purification of ADP-ribosylated nuclear proteins by covalent chromatography on dihydroxyboryl polyacrylamide beads and their characterization.

Nuclear proteins modified by mono or poly ADP-ribosylation were selectively isolated and purified by covalent chromatography on a dihydroxyboryl polyacrylamide bead column that specifically interacts with cis-diol-containing compounds. From rat liver nuclei that had been incubated with NAD+, histones and some nonhistone proteins were extracted with 0.25 M HCl. Approximately 60% of the ADP-ribose incorporated into 20% trichloroacetic acid-precipitable material was recovered in this extract. The ADP-ribosylated material was then isolated from the extract by covalent chromatography on a borate gel column and further purified by carboxymethylcellulose column chromatography. As judged by electrophoretic mobilities in various gel systems and by amino acid compositions, approximately 50% of the ADP-ribose recovered in the carboxymethylcellulose fractions was associated with several nonhistone proteins with molecular weights of 2-6 x 10(4), while 35% aand 15% were associated with histones H2B and H1, respectively. Since the average chain length of the polymer bound to any of these proteins was less than two ADP-ribos-l units, the percentage distribution reflects the number of ADP-ribosylated sites rather than the chain length.

Animals↗
PubMed1978

Novel enzyme from rat liver that cleaves an ADP-ribosyl histone linkage.

A novel enzyme that splits a bond between ADP-ribose and histone was discovered and partially purified from rat liver cytosol. The 105,000 X g supernatant of rat liver homogenate was precipitated by 45% saturated ammonium sulfate and then chromatographed on a DEAE-cellulose column. The enzyme activity was eluted in a single peak at about 0.2 M NaCl and clearly separated from poly(ADP-ribose) glycohydrolase which came out at 0.13 M NaCl. In contrast to the latter enzyme, this new enzyme catalyzed the spliting of a linkage between ADP-ribose and a protein portion in mono ADP-ribosylated histone H2B but little, if any, of the glycosidic ribosyl(1"-2') ribose bonds within poly(ADP-ribose). Analysis of the reaction product by paper chromatography and Dowex 1 column chromatography indicated that the split product contained the ADP-ribose moiety but was not exactly identical with ADP-ribose. Available evidence suggested that it was either an altered ADP-ribose molecule produced by a structural rearrangement or ADP-ribose itself linked to an unidentified compound. The enzyme had a pH optimum of about 6.0 and was inhibited by 80-90% in the presence of 5 mM ADP-ribose.

Animals↗
PubMed1979

Receptor-mediated transport of the hybrid protein ricin-diphtheria toxin fragment A with subsequent ADP-ribosylation of intracellular elongation factor II.

A hybrid protein of ricin and the enzymatically active fragment A of diphtheria toxin (toxin A) has been synthesized and purified. The diphtheria toxin A fragment of the hybrid protein is shown to enter the cytosol compartment of HeLa cells, its presence assayed by the fall of intracellular elongation factor II (EF-2) and the rise of ADP-ribosylated EF-2. Hybrid entrance to HeLa cells is blocked by lactose which blocks receptor-mediated entry of ricin but not by NH4Cl which blocks the transport of diphtheria toxin. It is concluded that the diphtheria toxin fragment A moiety of the hybrid enters the cell cytosol via the ricin receptor-mediated transport system. The kinetics of intracellular ADP-ribosylation of EF-2 by diphtheria toxin have also been studied. Ribosylation is preceded by a toxin dose-dependent lag period. The data suggest that the time constant responsible for the lag period is in the transport step. Models consistent with these data are discussed.

Adenosine Diphosphate Ribose↗
PubMed1977

The mechanism of ADP-ribosylation of elongation factor 2 catalyzed by fragment A from diphtheria toxin.

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 muM, 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.

Adenine↗
PubMed1979

ADP ribosylation of rat liver nucleosomal core histones.

When nucleosomal core histones were isolated from rat liver nuclei incubated with [14C]NAD+ and fractionated into the individual components (H2A, H2B, H3, and H4), [14C]adenosine diphosphate ribose (ADP-Rib) was found to be associated with all of them. However, while about 15% of the H2B molecules were modified, less than 2% of the other fractions contained radioactive ADP-Rib. The nucleotide attached to H2B was identified as a single monomer of ADP-Rib. On subjectint H2B to electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid, one single band of H2B with 5% less mobility than the unomdified control was obtained. The linkage between H2B and ADP-Rib was rapidly hydrolyzed with 0.1 N NaOH or with 1 M neutral hydroxylamine. Hydrolysis of ADP-ribosylated H2B with trypsin generated a single peptide linked to ADP-Rib, which corresponded to the sequence Pro-Glu-Pro-Ala-Lys. We were able to dansylate the NH2-terminal proline, which proved that the imino group of this amino acid was not substituted. These findings, together with the chemical properties of the linkage, which were typical of those of an ester-like bond, strongly suggest that the ADP-Rib residue was linked to the gamma-COOH group of the glutamic acid in position 2 of H2B.

Adenosine Diphosphate Sugars↗
PubMed1978

ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase.

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.

Adenosine Diphosphate Sugars↗
PubMed1979

Selection and characteristics of a Vibrio cholerae mutant lacking the A (ADP-ribosylating) portion of the cholera enterotoxin.

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.

Adenosine Diphosphate Sugars↗
PubMed1979

ADP-ribosylation of membrane proteins and activation of adenylate cyclase by cholera toxin in fat cell ghosts from euthyroid and hypothyroid rats.

Incubation of fat cell ghosts with activated cholera toxin, nucleoside triphosphate, cytosol, and NAD results in increased adenylate cyclase activity and the transfer of ADP-ribose to membrane proteins. The major ADP-ribose protein comigrates on sodium dodecyl sulfate-polyacrylamide gels with the putative GTP-binding protein of pigeon erythrocyte membranes (Mr 42 000), which is also ADP-ribosylated by cholera toxin. The treatment with cholera toxin enhances the stimulation of the fat cell membrane adenylate cyclase by GTP, but the stimulation by guanyl-5'-yl imidodiphosphate is unaltered. Subsequent stimulation of fat cell adenylate cyclase by 10 micrometers epinephrine is not particularly affected. These changes were qualititatively the same for membranes isolated from fat cells of hypothyroid rats. Although the cyclase of these membranes has a reduced response to epinephrine, guanyl-5'-yl imidodiphosphate or GTP, as compared to euthyroid rat fat cell membranes, the defect is not rectified by toxin treatment and cannot be explained by a deficiency in the cholera toxin target.

Adenosine Diphosphate Ribose↗
PubMed1979

ADP ribosylation of rat liver lysine-rich histone in vitro.

Purified rat liver nuclei were incubated with [14C]-NAD+ and the various nuclear protein fractions were separated. Forty per cent of the total radioactivity incorporated was associated with the histone fraction. Of this, about 50% was extracted with H1, in 0.5 N perchloric acid. When crude H1 was purified and fractionated into five different subfractions by chromatography on Bio-Rex 70, it was found that all the H1 subfractions contained radioactivity. This radioactive material was identified as oligomers of adenosine diphosphate ribose (ADP-Rib) with an average chain length which corresponded to trimers. The extent of the modification was dependent on the concentration of NAD+. About 60% of the H1 molecules were modified with a concentration of 1 mM NAD+. The presence of these oligomers of ADP-Rib introduced a large degree of microheterogeneity to H1 as detected by electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid. Bands of H1 with 10 to 20% less mobility than the unmodified H1 were present. Also, as a consequence of large content of ADP-Rib, the absorption maximum shifted from 275 to 259 nm. The half-life of the bond between the oligomers of ADP-Rib and H1 was about 3 min at 37 degrees C in the presence of 0.1 N NaOH, and 10 m1 were modified. The site of ADP ribosylation in the NH2-terminal half was localized in the tryptic peptide extending from the NH2-terminal end to lysine 15. The site of modification of the COOH-erminal half was localized in the tryptic peptide which contained the only glutamic acid residue in this fragment of H1...

Adenosine Diphosphate Sugars↗
PubMed1975

Natural occurrence of poly(ADP-ribosyl) histones in rat liver.

Poly(ADP-ribose) bound to histones has been isolated from rat liver. When [14C]ribose was administered intraperitoneally to rats at a dosage of 300-750 mug (100-250 muCi)/10o g, approximately 1% of the radioactivity was recovered in the acid (5% CLCCCOOH)-INSOLUBLE MATERIAL OF THE LIVER NUCLEI 2 HR AFTER INJECTION. Of the acid-insoluble radioactivity, 4.5-9% was extractable with 0.25 N HCL. Carboxymethyl-cellulose column chromatography of the HCl-extracted material revealed that the radioactivity cochromatographed with histone subfractions f1 and, to a lesser extent, f2 and f3. Part of the protein-bound radioactivity was rendered acid-soluble by treatment with either snake venom phosphodiesterase or neutral NH2OH. From the enzyme digest, 5'-AMP and psiADP-ribose [2'-(5"-phosphoribosyl)-5'-AMP] were recovered, while the NH2OH treatment yielded ADP-ribose monomer and, presumably, oligomer. These observations indicate that ADP-ribose is attached to histones in vivo and is present both as a monomer and a polymer.

Adenine↗
PubMed1977

The modification of nuclear proteins by ADP-ribosylation.

When incubated in vitro purified mouse nuclei incorporate NAD into poly(ADP-Rib). Analysis of the product on CsDl/urea gradients showed that a large proportion of the poly(ADP-Rib) was not attached to protein. The free poly(ADP-Rib) did not appear to arise through degradation and its chain length was significantly longer than the poly(ADP-Rib) attached to proteins. Fractionation of the proteins on hydroxyapatite revealed that tissue-specific modification of both the histones and non-histone proteins, had occurred. In the case of one protein species there was evidence for the existence of several forms with different numbers of ADP-Rib residues.

Animals↗