Purification and properties of glycohydrolase from calf thymus splitting ribose-ribose linkages of poly(adenosine diphosphate ribose).
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Adenosine Diphosphate↗
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The use of biotinylated poly(ADP-ribose) for studies on poly(ADP-ribose)-protein interaction.
Poly(ADP-ribose) is routinely detected by the use of radioactive polymers formed from labeled substrates. In this report a simple and time-saving method for the biotinylation and the detection of poly(ADP-ribose) on blots is described. The polymer modified by light-induced reaction with photobiotin was colorimetrically detected and quantified, using streptavidine-alkaline phosphatase conjugates. The separation of poly(ADP-ribose) chains on polyacrylamide gels was not affected by the biotinylation of the polymers. When biotinylated poly(ADP-ribose) was used to detect the poly(ADP-ribose) binding capability of proteins in ligand blots, the results were comparable to those obtained with poly([32P]ADP-ribose). Experiments with histones and rat liver nuclear proteins demonstrate that in studies on poly(ADP-ribose)-protein interaction, this method is applicable to the detection of poly(ADP-ribose) binding proteins.
Affinity Labels↗
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.
Adenosine Diphosphate Ribose↗
Mechanisms of poly(ADP-ribose) polymerase catalysis; mono-ADP-ribosylation of poly(ADP-ribose) polymerase at nanomolar concentrations of NAD.
Calf thymus and rat liver poly(ADP-ribose) polymerase enzymes, and the polymerase present in extracts of rat liver nuclei synthesize unstable mono-ADP-ribose protein adducts at 100 nM or lower NAD concentrations. The isolated enzyme-mono-ADP-ribose adduct hydrolyses to ADP-ribose and enzyme protein at pH values slightly above 7.0 indicating a continuous release of ADP-ribose from NAD through this enzyme-bound intermediate under physiological conditions. NH2OH at pH 7.0 hydrolyses the mono-ADP-ribose enzyme adduct. Desamino NAD and some other homologs at nanomolar concentrations act as 'forward' activators of the initiating mono-ADP-ribosylation reaction. These NAD analogs at micromolar concentrations do not affect polymer formation that takes place at micromolar NAD concentrations. Benzamides at nanomolar concentrations also activate mono-ADP-ribosylation of the enzyme, but at higher concentrations inhibit elongation at micromolar NAD as substrate. In nuclei, the enzyme molecule extensively auto-ADP-ribosylates itself, whereas histones are trans-ADP-ribosylated to a much lower extent. The unstable mono-ADP-ribose enzyme adduct represents an initiator intermediate in poly ADP-ribosylation.
ADP Ribose Transferases↗
Detection of poly(ADP-ribose) polymerase and its reaction product poly(ADP-ribose) by immunocytochemistry.
Poly(ADP-ribose) polymerase catalyses the formation of ADP-ribose polymers covalently attached to various nuclear proteins, using NAD+ as substrate. The activity of this enzyme is strongly stimulated upon binding to DNA single or double strand breaks. Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage and is thought to be involved in DNA repair, genetic recombination, apoptosis and other processes during which DNA strand breaks are formed. In recent years we and others have established cell culture systems with altered poly(ADP-ribose) polymerase activity. Here we describe immunocytochemistry protocols based on the use of antibodies against the DNA-binding domain of human poly(ADP-ribose) polymerase and against its reaction product poly(ADP-ribose). These protocols allow for the convenient mass screening of cell transfectants with overexpression of poly(ADP-ribose) polymerase or of a dominant-negative mutant for this enzyme, i.e. the DNA-binding domain. In addition, the immunocytochemical detection of poly(ADP-ribose) allows screening for cells with altered enzyme activity.
Animals↗
Poly(ADP-ribose) synthetase, a main acceptor of poly(ADP-ribose) in isolated nuclei.
Poly(ADP-ribose) synthetase was identified as the main acceptor of this polymer produced in isolated nuclei of rat liver. When the nuclei were incubated with [32P]NAD at a limited concentration (2.4 microM) and for a brief period (10 s), a protein with Mr = 110,000 was predominantly poly(ADP-ribosyl)ated, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The modification of this protein increased upon longer incubations or at higher NAD concentrations, and induced a marked increase in the apparent molecular weight. A comparison with poly(ADP-ribose) synthetase (Mr = 110,000) of rat liver under various conditions suggested that the increase in the molecular weight of the acceptor resembled that of the synthetase undergoing multiple auto-poly(ADP-ribosyl)ation. This interpretation was further supported by the following observations: 1) [32P]poly(ADP-ribose) attached to the acceptor co-eluted with the synthetase activity from a hydroxyapatite column; 2) the [32P]poly(ADP-ribose).acceptor complex isolated on the column was converted to a very large complex by further incubation with NAD; and 3) a group of large poly(ADP-ribose).acceptor complexes were reduced to a single molecular species with Mr = 110,000 by extensive digestion with poly(ADP-ribose) glycohydrolase. These findings altogether suggested that poly(ADP-ribose) synthesized in isolated nuclei was principally bound to the synthetase itself.
Animals↗
D-ribose and deoxy-D-ribose induce apoptosis in human quiescent peripheral blood mononuclear cells.
In previous papers we reported that D(-)-ribose and 2-deoxy-D-ribose, which rank at the top among reducing sugars, kill a variety of human and animal cells and cell lines. Here we demonstrate that these two sugars induce apoptosis in human quiescent peripheral blood mononuclear cells which are relatively insensitive to apoptosis. Apoptosis was assessed by morphological changes, DNA fragmentation by agarose gel electrophoresis and the appearance of an hypodiploid peak by flow cytometry. 2-deoxy-D-ribose was more potent than D(-)-ribose and apoptosis was evident from 48 hours of culture onwards. 2-deoxy-D-ribose-induced apoptosis was inhibited by N-acetyl-L-cysteine, suggesting that glutathione metabolism and/or oxidative stress are involved in this type of apoptosis. Thus, D(-)-ribose and 2-deoxy-D-ribose can be useful tools to study the cellular and molecular events of apoptosis in human quiescent lymphocytes.
Acetylcysteine↗
Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair.
We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.
Animals↗
Changes in poly(adenosine diphosphate-ribose) and poly(adenosine diphosphate-ribose) polymerase in synchronous HeLa cells.
An antibody has been prepared which is highly specific for poly(adenosine diphosphate-ribose). Neither poly(A), DNA, nor a variety of adenine-containing nucleosides or nucleotides were effective in competing with poly(ADP-ribose) for binding to the antibody. Of all compounds tested, only adenosine diphosphate-ribose competed for binding to the antibody. Unlabeled poly(adenosine diphosphate-ribose) was about 10 000 times more effective in competing with labeled polymer for antibody binding than was adenosine diphosphate-ribose. Using the antibody, the amount of poly(adenosine diphosphate-ribose) was found to increase from early S phase to a peak at mid S with a second, even larger increase seen at the S-G2 transition point in synchronously dividing HeLa cells. Pulse labeling of the polymer with [2-3H]adenosine was also maximal at the same time points. Changes in the levels of poly(adenosine diphosphate-ribose) polymerase activity measured in isolated nuclei coincided with the changes in amounts of polymer present in intact cells during progression from S phase into G2.
Adenine Nucleotides↗
Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins.
Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.
Animals↗
Poly(ADP-ribosylation) at successive stages of rooster spermatogenesis. Levels of polymeric ADP-ribose in vivo and poly(ADP-ribose) polymerase activity and turnover of ADP-ribosyl residues in vitro.
Rooster testis cells were separated by sedimentation at unit gravity and the in vivo levels of polymeric ADP-ribose were determined both in intact cells and isolated nuclei by fluorescence methods. Poly(ADP-ribose) polymerase activity was assayed after cell permeabilization or after isolation of nuclei. The turnover of ADP-ribosyl residues was determined in isolated nuclei using benzamide. The content of poly(ADP-ribose), the poly(ADP-ribose) polymerase activity, and the turnover of ADP-ribosyl residues, decreased during the differentiation of the germinal cell line, especially at the end of spermiogenesis. Treatment of cells with 1 mM dimethyl sulfate for 1 h resulted in a marked stimulation of poly(ADP-ribose) polymerase activity in meiotic and premeiotic cells and also in round and late spermatids. The enzymatic activity was not detected and could not be induced in mature spermatozoa. These cells, however, still contained polymeric ADP-ribose with a 2% of branched form.
Adenosine Diphosphate Ribose↗
Novel hydrolysis-resistant analogues of cyclic ADP-ribose: modification of the "northern" ribose and calcium release activity.
Three novel analogues modified in the "northern" ribose (ribose linked to N1 of adenine) of the Ca(2+) mobilizing second messenger cyclic adenosine diphosphoribose, termed 2"-NH(2)-cyclic adenosine diphosphoribose, cyclic adenosine diphospho-carbocyclic-ribose, and 8-NH(2)-cyclic adenosine diphospho-carbocyclic-ribose, were synthesized (chemoenzymatically and by total synthesis) and spectroscopically characterized, and the pK(a) values for the 6-amino/imino transition were determined in two cases. The biological activity of these analogues was determined in permeabilized human Jurkat T-lymphocytes. 2"-NH(2)-cyclic adenosine diphosphoribose mediated Ca(2+) release was slightly more potent than that of the endogenous cyclic adenosine diphosphoribose in terms of the concentration-reponse relationship. Both compounds released Ca(2+) from the same intracellular Ca(2+) pool. In addition, the control compound 2"-NH(2)-adenosine diphosphoribose was almost without effect. In contrast, only at much higher concentrations (> or =50 microM) did the "northern" carbocyclic analogue, cyclic adenosine diphospho-carbocyclic-ribose, significantly release Ca(2+) from permeabilized T cells, whereas the previously reported "southern" carbocyclic analogue, cyclic aristeromycin diphosphoribose, was slightly more active than the endogenous cyclic adenosine diphosphoribose. Likewise, 8-NH(2)-cyclic adenosine diphospho-carbocyclic-ribose, expected to antagonize Ca(2+) release as demonstrated previously for 8-NH(2)-cyclic adenosine diphosphoribose, did not inhibit cyclic adenosine diphosphoribose mediated Ca(2+) release. This indicates that the 2"-NH(2)-group substitutes well for the 2"-OH-group it replaces; it may be oriented toward the outside of the putative cyclic adenosine diphosphoribose receptor binding domain and/or it can potentially also engage in H bonding interactions with residues of that domain. In sharp contrast to this, replacement of the endocyclic furanose oxygen atom by CH(2) in a carbocyclic system obviously interferes with a crucial element of interaction between cyclic adenosine diphosphoribose and its receptor in T-lymphocytes.
Adenosine Diphosphate Ribose↗
UV irradiation inhibits ABC transporters via generation of ADP-ribose by concerted action of poly(ADP-ribose) polymerase-1 and glycohydrolase.
ATP-binding cassette (ABC) transporters are involved in the transport of multiple substrates across cellular membranes, including metabolites, proteins, and drugs. Employing a functional fluorochrome export assay, we found that UVB irradiation strongly inhibits the activity of ABC transporters. Specific inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) restored the function of ABC transporters in UVB-irradiated cells, and PARP-1-deficient cells did not undergo UVB-induced membrane transport inhibition. These data suggest that PARP-1 activation is necessary for ABC transporter functional downregulation. The hydrolysis of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG) was also required, since specific PARG inhibitors, which limit the production of ADP-ribose molecules, restored the function of ABC transporters. Furthermore, ADP-ribose molecules potently inhibited the activity of the ABC transporter P-glycoprotein. Hence, poly(ADP-ribose) metabolism appears to play a novel role in the regulation of ABC transporters.
ATP-Binding Cassette Transporters↗
Chain length analysis of ADP-ribose polymers generated by poly(ADP-ribose) polymerase (PARP) as a function of beta-NAD+ and enzyme concentrations.
Bireactant autopoly(ADP-ribosyl)ation of poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30) was carried out by using either increasing concentrations of beta-NAD+ (donor substrate) at a fixed protein concentration or increasing concentrations of PARP (acceptor substrate) at a fixed beta-NAD+ concentration. The [32P]ADP-ribose polymers synthesized were chemically detached from PARP by alkaline hydrolysis of the monoester bond between the carboxylate moiety of Glu and the polymer. Nucleic acid-like polymers were then analyzed by high-resolution polyacrylamide gel electrophoresis and autoradiography. The ADP-ribose chain lengths observed displayed substrate concentration-dependent elongation from 0.2 microM to 2 mM beta-NAD+. Similar results were observed at fixed concentrations of 4.5, 9, 18, 27, and 36 nM PARP. Therefore, we conclude that the concentration of the ADP-ribose donor substrate determines the average chain length of the polymer synthesized. In contrast, the polymer size was unaltered when the concentration of PARP was varied from 4.5 to 18 nM at a fixed beta-NAD+ concentration. However, when PARP concentrations > 18 nM were used, the total amount of monomeric ADP-ribose produced was noticeably less. Therefore, we conclude that high concentrations of PARP lead to acceptor substrate inhibition at the level of the ADP-ribose chain initiation reaction.
Adenosine Diphosphate Ribose↗
3'-Deoxy-NAD+ as a substrate for poly(ADP-ribose)polymerase and the reaction mechanism of poly(ADP-ribose) elongation.
Adenyl-32P-Labeled 3'-deoxy-NAD+ was utilized as a substrate by pure DNA-dependent poly(ADP-ribose)polymerase (EC 2.4.2.30) from calf thymus in the automodification reaction with an apparent Km of 20 microM and a Vmax of 80 nmol/min/mg of protein. Analysis by lithium lauryl sulfate-polyacrylamide gel electrophoresis revealed a single 32P-labeled protein of 116-kDa which comigrated with automodified enzyme. Addition of increasing amounts of histone H1 up to a concentration of 15 micrograms/ml stimulated the synthesis of protein-bound polymers of 3'-deoxy-ADP-ribose. However, the average polymer size was equal to 2 in the presence and 4 in the absence of histone H1, respectively. The synthesis of protein-bound oligomers of 3'-deoxy-ADP-ribose was inhibited by the polymerase inhibitors benzamide, nicotinamide, thymidine, and NaCl. A pulse labeling of polymer synthesis with 40 microM [32P]3'-deoxy-NAD+ either in the presence or absence of 15 micrograms/ml of histone H1, followed by a chase with 1 mM [3H]NAD+, was used to determine the mechanism of poly(ADP-ribose) elongation. Following enzyme digestion of these polymers with phosphodiesterase, it was found that 52 and 24% of the total 32P radiolabel was associated with the 3'-deoxy-AMP termini of the polymers synthesized in the pulse reactions, in the presence or absence of histone H1, respectively. In contrast, less than 10% of the total radioactivity was associated with 3'-deoxy-AMP in the product of the chase reactions. These results are consistent with the conclusion that the initially attached residue of 3'-deoxy-ADP-ribose to either the polymerase or histone H1, is elongated by the "protein-distal" addition of ADP-ribose residues to the AMP terminus of the growing polymer chain.
Animals↗
Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts.
Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A+) mRNA detection by hybridization to [3H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21,000-120,000 range. The Mr 120,000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21,000 protein acceptor is abundant in PMS and a Mr 34,000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.
Animals↗
Effect of DNA on poly (ADP-ribose) glycohydrolase and the degradation of histone H1-poly (ADP-ribose) complex from HeLa cell nuclei.
A poly(ADP-ribose)-H1 histone complex has been isolated from HeLa cell nuclei incubated with NAD. The rate of poly(ADP-ribose) glycohydrolase catalyzed hydrolysis of the polymer in the complex is only 1/9 that of free poly(ADP-ribose), indicating that the polymer is in a protected environment within the complex. Comparison of the rate of hydrolysis of free poly(ADP-ribose) in the presence or absence of H1 to that in the complex synthesized de novo indicates a specific mode of packaging of the complex. This is further indicated by the fact that alkaline dissociation of the complex followed by neutralization markedly exposes the associated poly(ADP-ribose) to the glycohydrolase. The complex also partially unfolds when it binds to DNA as evidenced by a 2-fold increase in the rate of glycolytic cleavage of poly(ADP-ribose). This effect of DNA is not due to a stimulation of the glycohydrolase per se since hydrolysis of free polymer by the enzyme is strongly inhibited by DNA, especially single-stranded DNA. Inhibition of glycohydrolase by DNA results from the binding of the enzyme to DNA and conditions which decrease this binding (increased ionic strength or addition of histone H1 which competes for DNA binding) relieve the DNA inhibition.
Cell Nucleus↗
Poly(ADP-ribose) accumulation and enhancement of postischemic brain damage in 110-kDa poly(ADP-ribose) glycohydrolase null mice.
Poly(ADP-ribose) (PAR) is a polymer synthesized by poly(ADP-ribose) polymerases (PARPs) and metabolized into free adenosine diphosphate (ADP)-ribose units by poly(ADP-ribose) glycohydrolase (PARG). Perturbations in PAR synthesis have been shown to play a key role in brain disorders including postischemic brain damage. A single parg gene but two PARG isoforms (110 and 60 kDa) have been detected in mouse cells. Complete suppression of parg gene causes early embryonic lethality, whereas mice selectively lacking the 110 kDa PARG isoform (PARG(110)(-/-)) develop normally. We used PARG(110)(-/-) mice to evaluate the importance of PAR catabolism to postischemic brain damage. Poly(ADP-ribose) contents were higher in the brain tissue of PARG(110)(-/-) than PARG(110)(+/+) mice, both under basal conditions and after PARP activation. Distal middle cerebral artery occlusion caused higher increase of brain PAR levels and larger infarct volumes in PARG(110)(-/-) mice than in wild-type counterparts. Of note, the brain of PARG(110)(-/-) mice showed reduced heat-shock protein (HSP)-70 and increased cyclooxygenase-2 expression under both control and ischemic conditions. No differences were detected in brain expression/activation of procaspase-3, PARP-1, Akt, HSP-25 and interleukin-1beta. Our findings show that PAR accumulation worsens ischemic brain injury, and highlight the therapeutic potential of strategies capable of maintaining PAR homeostasis.
Adenosine Triphosphate↗