Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pertussis toxin (islet-activating protein) activates adenylate cyclase in susceptible cells by ADP-ribosylating an inhibitory component of the cyclase system. This toxin, assayed in a cell-free system in the presence of high concentrations of thiol, catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. This NAD glycohydrolase activity co-chromatographed on Sephacryl G-200 in 6.5 M urea, pH 3.2, 0.1 M glycine with the ADP-ribosyltransferase activity of the toxin, as monitored by the transfer of [32P]ADP-ribose from [32P]NAD to a 41,000-Da protein in NG108-15 neuroblastoma X glioma hybrid cells. In the absence of thiol, the native holotoxin was enzymatically inactive. Following addition of 250 mM dithiothreitol to the assay, maximal enzymatic activity was evident after a delay of approximately 1 h; with 20 mM thiol, the delay was longer. The Km for NAD with the fully activated enzyme was 25 microM; the Km did not appear to vary with the extent of activation. Thiol was necessary in a cell-free system to demonstrate NAD glycohydrolase activity. When extensively washed membranes were used as a source of 41,000-Da substrate, thiol was necessary to observe ADP-ribosylation in some cases (human erythrocytes) and significantly stimulated activity in others (NG108-15 cells). In contrast to the bacterial toxins choleragen and Escherichia coli heat-labile enterotoxin that ADP-ribosylate stimulatory components of the cyclase system, pertussis toxin did not transfer ADP-ribose to low molecular weight guanidino compounds, such as arginine or agmatine.
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PMID:Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein). 631 27

Oxidized nicotinamide adenine dinucleotide (NAD+) in cytosol may interact with renal brush-border membranes (BBM) and inhibit BBM phosphate transport. The possible mechanism of interaction was investigated in the present study. Incubation of BBM with [adenine-3H]NAD+ led to acid-stable binding of 3H to the BBM, in contrast there was no binding of 14C when [carbonyl-14C]NAD+ was used. The data are consistent with an ADP-ribosylation mechanism involving transfer of ADP-ribose from NAD+ to BBM. This was confirmed by using [adenylate-32P]NAD+ and by the release of bound 32P in the form of 5'-[32P]AMP when the BBM were treated with snake venom phosphodiesterase. After gradient centrifugation of BBM the ADP-ribosyltransferase was recovered at the same density as known BBM enzymes, indicating that ADP-ribosyltransferase is an intrinsic BBM component and not a contaminant. These findings indicate that cytosolic NAD+ may be used for ADP-ribosylation of BBM proteins and that this may be a mechanism for regulating the BBM phosphate transport system.
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PMID:NAD+-dependent ADP-ribosyltransferase in renal brush-border membranes. 631 20

Chromatin-bound ADP-ribosyltransferase from adult hen liver nuclei was purified to a homogeneous state through salt extraction, gel filtration, hydroxyapatite, phenyl-Sepharose, Cm-cellulose, and DNA-Sepharose. The ADP-ribosyltransferase has a pH optimum at 9.0 and does not require DNA for reaction. The purified enzyme has a molecular weight of 27,500 +/- 500. Agmatine sulfate, arginine methyl ester, histones, and casein proved to be effective acceptors for the ADP-ribose molecule. Among histones, H3 was most active, followed by H2a, H4, and H2b, in that order, the lowest activity seen with H1. With all the acceptors tested, the rate of nicotinamide release was in excess of the ADP-ribosylation. However, changes in the ratio of nicotinamide release to ADP-ribosylation seemed to depend on concentrations of the acceptor used. ADP-ribose-whole histones X adducts formed by ADP-ribosyltransferase served as initiators for poly(ADP-ribose) synthesis when these adducts were incubated in the presence of NAD, DNA, Mg2+, and the purified poly(ADP-ribose) synthetase, in which poly(ADP-ribose) formation can occur.
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PMID:ADP-ribosyltransferase from hen liver nuclei. Purification and characterization. 631 19

ADP-ribosyltransferase activity has been characterized in free messenger ribonucleoprotein particles (mRNP) from mouse plasmacytoma cells. This enzymatic activity appears to be associated with the free mRNP and not due to nuclear contamination. The enzyme activity is not stimulated by added DNA or histone H1 and represents 34 per cent of the total cellular ADP-ribosyltransferase activity while the DNA contamination in free mRNP is less than 4 per cent of the total cellular DNA. Moreover, the ADP-ribosyltransferase specific activity per mg of DNA is about 75-fold higher in free mRNP than in the nuclei. During CsCl gradient centrifugation of the cytoplasmic fraction, the ADP-ribosylated material separates out at a buoyant density similar to that of free mRNP. This ADP-ribosyltransferase activity is inhibited by thymidine, nicotinamide and 3-aminobenzamide, while it is highly stimulated by exogenous pancreatic RNase. The in vitro synthesized acid insoluble material is rendered partly soluble by treatment by a proteolytic enzyme or by snake venom phosphodiesterase resulting in phosphoribosyl-AMP formation: the pancreatic RNase does not solubilize this material. Several ADP-ribosylated proteins are detected by lithium dodecylsulfate gel electrophoresis. Such an ADP-ribosyltransferase activity has also been detected in free mRNP from rat liver. It is suggested that this ADP-ribosylation of specific free mRNP proteins may be associated with free mRNP structure and/or with some chemical covalent type of modification rendering mRNA available for translation.
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PMID:Adenosine diphosphate ribosyltransferase and protein acceptors associated with cytoplasmic free messenger ribonucleoprotein particles. 632 87

Cholera toxin catalyzed the ADP-ribosylation of a single plasma membrane protein (Mr 55 000) of both RL-PR-C rat hepatocytes and purified rat liver plasma membranes. Labeling of this protein from nicotinamide [2,8-3H]adenine dinucleotide was competitively inhibited by free arginine, but by no other amino acid tested, including lysine. The same protein was ADP-ribosylated from NAD+ endogenously, i.e., in the absence of toxin. This process was, however, not competitively inhibited by added arginine nor by any other amino acid tested lysine. Free ADP-ribose, even in 50-fold molar excess over the nicotinamide [2,8-3H]adenine dinucleotide substrate, did not reduce (by isotope dilution) the endogenous or cholera toxin-catalyzed labeling of the 55 000 dalton membrane protein. It is likely, therefore, that hepatocyte plasma membranes contain an ADP-ribosyltransferase, with a mechanism similar to that of the A subunit of cholera toxin, in that both transfer ADP-ribose to the same membrane protein and in that neither apparently produce free ADP-ribose as an intermediate. It is also clear that the acceptor residue in the 55 000 dalton protein is different for each process. Cholera toxin-catalyzed and endogenous transfer of ADP-ribose to the hepatocyte plasma membrane protein, in contrast to a pigeon erythrocyte system, required no cytosolic factors. The results indicate that ADP-ribosylation in cloned differentiated rat hepatocytes differs from that in pigeon erythrocytes in that the acceptor protein is larger (55 000 compared to 42 000 daltons), cytosolic factors are not required and transfer of ADP-ribose to the acceptor protein occurs endogenously.
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PMID:Endogenous and cholera toxin-catalyzed ADP-ribosylation of a plasma membrane protein by RL-PR-C cloned rat hepatocytes. 722 28

We have recently described that poly(ADP-ribosyl)-polymerase (PARP) inhibitors rescue U937 cells from apoptosis induced by 1 mM H2O2 oxidative stress; PARP activation leads to a reversible drop in NAD level, which could be blocked by PARP inhibitors (Nos-seri et al., 1994, Exp. Cell Res. 212, 367-373). A phenotypic variant of U937 is characterized by a lower basal NAD level (low NAD, LN U937, as opposed to the original high NAD, HN U937). In LN cells treatment with 1 mM H2O2, although activating PARP, does not lower NAD concentration; puzzlingly, PARP inhibitors increase (instead of decreasing, as occurs in HN cells) the extent of stress-induced apoptosis, leading to a reduced cell survival. NAD concentration could be increased in LN cells by adding nicotinamide (5-and 25-fold increase) to the culture medium. These cells (LN+) behaved as HN U937: oxidative stress induced a NAD drop, the extent of which is dependent on the cells' basal NAD level; moreover, PARP inhibitors could rescue LN+ cells from peroxide-induced apoptosis. H2O2-induced apoptosis is not triggered by NAD depletion, but instead it takes place only when NAD levels have been preserved or have recovered: on HN U937, peroxide doses (5 and 10 mM) which lead to necrosis induce an irreversible NAD drop, whereas apoptosis occurs only at lower doses, where NAD depletion is reversible; on LN cells NAD levels do not drop even upon 10 mM H2O2 treatment, and these cells die only by apoptosis; moreover, in HN cells apoptosis is not detectable until 8 h posttreatment, when NAD levels recover, whereas in LN cells, where NAD is always present, apoptosis begins to take place as early as 3 h after stress.
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PMID:Different basal NAD levels determine opposite effects of poly(ADP-ribosyl)polymerase inhibitors on H2O2-induced apoptosis. 749 46

HN and LN are two phenotypic variants of the U937 monocytic cell line which differ in their basal NAD content; they respond in an opposite way to oxidative stress in the presence of the poly(ADP-ribosyl)polymerase (PARP) inhibitors 3-aminobenzamide (3ABA) and nicotinamide (NA): the inhibitors protect HN cells from stress-induced apoptosis, while they enhance it on LN cells (Coppola et al., 1995, Exp. Cell Res. 221, 462-469). These opposite effects are due to two overlapping and contrasting phenomena occurring in LN cells, as shown by the bi-modal response of stressed LN cells to increasing 3ABA doses. Indeed H2O2-induced apoptosis is enhanced only at high 3ABA concentrations (i.e., sufficient to inhibit also mono-ADP-ribosylations); lower 3ABA concentrations, which specifically inhibit PARP, also protect LN U937 from stress-induced apoptosis. Unlike HN U937, H2O2-induced apoptosis in LN cells is accompanied by cell blebbing. High 3ABA doses strongly enhance blebbing, leading to cellular fragmentation. Blebbing could be blocked by interfering with actin polymerization with cytochalasin B and D: this eliminated the increase in apoptosis due to 3ABA, suggesting that it is indeed the consequence of excess blebbing. This is supported by the unusual finding that in U937 LN stressed in the presence of 3ABA or NA, blebbing, usually a late event in apoptosis, may even precede its onset.
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PMID:The increase in H2O2-induced apoptosis by ADP-ribosylation inhibitors is related to cell blebbing. 749 47

An NAD+:cysteine ADP-ribosyltransferase activity was purified from bovine erythrocytes on the assumption that, like pertussis toxin, the enzyme would exhibit a cysteine-dependent NAD+ glycohydrolase activity. A three-step purification procedure was developed involving (1) precipitation with 40% (NH4)2SO4, (2) binding to a cysteine-Sepharose affinity column, and (3) binding to an NAD+ affinity column. PAGE showed a single band of M(r) 45,000. The enzyme had been purified 47,000-fold and had a specific activity of 1900 nmol nicotinamide released/min per mg. A study of the kinetic properties of this enzyme showed saturation kinetics for cysteine (Km = 4.0 mM). The ability of this enzyme to ADP-ribosylate protein was investigated using re-sealed inverted bovine erythrocyte ghosts. Incubation of the purified enzyme with erythrocyte ghosts and [adenylate-32P]NAD+ led to the enhanced dose-dependent labelling of several proteins, a doublet of high M(r) and proteins of M(r) 60,000, 55,000 and 29,000, identified by autoradiography of separated proteins on SDS/PAGE. The enzyme-catalysed labelling of the major component at M(r) 55,000 was blocked by pre-treatment of the erythrocyte ghosts with N-ethymaleimide, a sulphydryl alkylating agent, and the label was released by mercuric ion, but not by hydroxylamine. These experiments suggested that a cysteine residue on the target protein had been mono-ADP-ribosylated. This supposition was further supported by identification of the mercf1p4ion-released radiolabelled product as ADP-ribose by HPLC, and the observation that free ADP-ribose was unable to modify the membrane target protein directly.
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PMID:The purification of a cysteine-dependent NAD+ glycohydrolase activity from bovine erythrocytes and evidence that it exhibits a novel ADP-ribosyltransferase activity. 757 29

Poly(ADPR) polymerase (PARP; EC 2.4.2.30) is a nuclear enzyme, which, when activated by oxygen- and nitrogen-radical-induced DNA strand breaks, transfers ADP ribose units to nuclear proteins and initiates apoptosis by depletion of cellular NAD and ATP pools. The present study investigates whether the oxidative stress-dependent activation of PARP plays a role in the etiopathogenesis of arthritis. The antiarthritic reactivity of the biogenic PARP inhibitor nicotinamide was tested in DBA/1 x B10A(4R) mice suffering from potassium peroxochromate-induced arthritis. Daily doses of 4 mmol/kg of NA suppressed the arthritis by 35% and inhibited the phagocytic generation of reactive oxygen species, which increases sixfold during the development of arthritis. The onset, progression, and remission of arthritis correlated positively to the phorbolester-activated respiratory burst of neutrophils and monocytes, and a dose-dependent inhibition of NADPH oxidase activity was determined with human phagocytes. Our data support the hypothesis that oxidative stress-induced alterations in cellular signal transduction pathways play a pivotal role in the development of arthritis, which can be suppressed by the simultaneous inhibition of poly(ADPR) polymerase and NADPH oxidase.
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PMID:Modulation of inflammatory arthritis by inhibition of poly(ADP ribose) polymerase. 762 65

Exposure to hydrogen peroxide (H2O2) decreases phosphatidylcholine (PC) synthesis in rabbit type II pneumocytes. Activation of poly(ADP-ribose) polymerase (PARP) may play a role in this process. Exposure of type II pneumocytes to H2O2 resulted in a 53% decrease in the rate of incorporation of [3H]choline into PC (P < 0.001). Cell NAD and ATP levels were decreased by 52% (P < 0.001) and 39% (P < 0.01), respectively, without significant changes in cell viability. Exposure to H2O2 also resulted in a 52% (P < 0.05) increase in the activity of PARP. Preincubation of type II cells with inhibitors of PARP (nicotinamide; 3-aminobenzamide) before H2O2 exposure prevented the increase in PARP activity, and blocked the decreases in ATP, NAD, and rate of PC synthesis. These results suggest that the energy depletion associated with activation of PARP contributes to the effects of oxidant stress on type II cell metabolic function and may be ameliorated by pharmacological agents in vitro.
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PMID:Inhibition of poly(ADP-ribose) polymerase preserves surfactant synthesis after hydrogen peroxide exposure. 763 15


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