Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation.Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment.PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome.Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as providing the basis for selection of the appropriate genetic model organisms to study the physiology of the specific human PARP proteins.     

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites

Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased V_max and decreased the K_m for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family members.     

Structure and function of the ARH family of ADP-ribosyl-acceptor hydrolases

ADP-ribosylation is a post-translational protein modification, in which ADP-ribose is transferred from nicotinamide adenine dinucleotide (NAD⁺) to specific acceptors, thereby altering their activities. The ADP-ribose transfer reactions are divided into mono- and poly-(ADP-ribosyl)ation. Cellular ADP-ribosylation levels are tightly regulated by enzymes that transfer ADP-ribose to acceptor proteins (e.g., ADP-ribosyltransferases, poly-(ADP-ribose) polymerases (PARP)) and those that cleave the linkage between ADP-ribose and acceptor (e.g., ADP-ribosyl-acceptor hydrolases (ARH), poly-(ADP-ribose) glycohydrolases (PARG)), thereby constituting an ADP-ribosylation cycle. This review summarizes current findings related to the ARH family of proteins. This family comprises three members (ARH1-3) with similar size (39 kDa) and amino acid sequence. ARH1 catalyzes the hydrolysis of the N-glycosidic bond of mono-(ADP-ribosyl)ated arginine. ARH3 hydrolyzes poly-(ADP-ribose) (PAR) and O-acetyl-ADP-ribose. The different substrate specificities of ARH1 and ARH3 contribute to their unique roles in the cell. Based on a phenotype analysis of ARH1^−/− and ARH3^−/− mice, ARH1 is involved in the action by bacterial toxins as well as in tumorigenesis. ARH3 participates in the degradation of PAR that is synthesized by PARP1 in response to oxidative stress-induced DNA damage; this hydrolytic reaction suppresses PAR-mediated cell death, a pathway termed parthanatos.     

Immunochemical detection of guanine nucleotide binding proteins mono-ADP-ribosylated by bacterial toxins

Rabbits immunized with ADP-ribose chemically conjugated to carrier proteins developed antibodies reactive against guanine nucleotide binding proteins (G proteins) that had been mono-ADP-ribosylated by bacterial toxins. Antibody reactivity on immunoblots was strictly dependent on incubation of substrate proteins with both toxin and NAD and was quantitatively related to the extent of ADP-ribosylation. Gi, Go, and transducin (ADP-ribosylated by pertussis toxin) and elongation factor II (EF-II) (ADP-ribosylated by pseudomonas exotoxin) all reacted with ADP-ribose antibodies. ADP-ribose antibodies detected the ADP-ribosylation of an approximately 40-kilodalton (kDa) membrane protein related to Gi in intact human neutrophils incubated with pertussis toxin and the ADP-ribosylation of an approximately 90-kDa cytosolic protein, presumably EF-II, in intact HUT-102 cells incubated with pseudomonas exotoxin. ADP-ribose antibodies represent a novel tool for the identification and study of G proteins and other substrates for bacterial toxin ADP-ribosylation.     

Activation of NUDT5, an ADP-ribose pyrophosphatase, by nitric oxide-mediated ADP-ribosylation

The ADP-ribose (ADPR) pyrophosphatase (ADPRase) NUDT5, a member of a superfamily of Nudix hydrolases, hydrolyzes ADP-ribose (ADPR) to AMP and ribose 5'-phosphate. Nitric oxide (NO) enhances nonenzymatic ADP-ribosylation of proteins such as beta-actin and glyceraldehydes 3-phosphate dehydrogenase in the presence of free ADPR, suggesting a possibility that NUDT5 could also be ADP-ribosylated by its substrate, ADPR. Here, we show that NO stimulates nonenzymatic ADP-ribosylation of NUDT5 using ADP-ribose and consequently activates its ADPRase activity. We found that ADPRase activity in J774 macrophage cells is increased by the treatment with SNP, an exogenous NO generator or TNF-alpha/IFN-gamma, endogenous NO inducers. Anti-NUDT5 antibody pulled down most of the ADPRase activity increased by NO, indicating that the ADPRase regulated by NO is NUDT5. Using recombinant human NUDT5, we also demonstrated that the increase of ADPRase activity is mediated via ADP-ribosylation at cysteine residue(s) in the presence of reductant. This result suggests that NO activates NUDT5 through ADP-ribosylation at cysteine residues of the enzyme in macrophages.     

Inhibition of prolyl hydroxylase by poly(ADP-ribose) and phosphoribosyl-AMP. Possible role of ADP-ribosylation in intracellular prolyl hydroxylase regulation

Poly(ADP-ribose) prepared by incubating NAD+ with rat liver nuclei inhibited the hydroxylation reaction catalyzed by purified prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) in vitro. Near complete inhibition of the enzyme was seen in the presence of 6 nM (ADP-Rib)18 with a Ki(app) of 1.5 nM. The monomer unit of poly(ADP-ribose), adenosine diphosphoribose (ADP-Rib), was found to be a weak inhibitor. On the other hand, poly(ADP-ribose)-derived phosphoribosyl-AMP (PRib-AMP) and its dephosphorylated product, ribosyl-ribosyl-adenine (Rib-RibA), inhibited the enzyme in nanomolar concentrations (Ki(app) 16.25 nM). The order of inhibition was (ADP-Rib)18 greater than PRib-AMP, Rib-RibA much greater than ADP-Rib. These results suggested that the 1"----2' ribosyl-ribosyl moiety in these compounds was involved in the inhibition of the enzyme. The possibility that intracellular prolyl hydroxylase is regulated by the involvement of ADP-ribosylation reactions was examined in confluent cultures of skin fibroblast treated with 20 mM lactate. The activity of prolyl hydroxylase was stimulated by 145% over that of untreated cultures. In the lactate-treated cells, the level of NAD+ was lowered and the total ADP-ribosylation of cellular proteins reduced by 40%. These observations imply that the lactate-induced activation of cellular prolyl hydroxylase is mediated by a reduction in ADP-ribosylation and that the synthesis and degradation of ADP-ribose moiety(ies) may possibly regulate prolyl hydroxylase activity in vivo.     

Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair.     

New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs

Poly(ADP-ribose) polymerases (PARPs) are enzymes that transfer ADP-ribose groups to target proteins and thereby affect various nuclear and cytoplasmic processes. The activity of PARP family members, such as PARP1 and PARP2, is tied to cellular signalling pathways, and through poly(ADP-ribosyl)ation (PARylation) they ultimately promote changes in gene expression, RNA and protein abundance, and the location and activity of proteins that mediate signalling responses. PARPs act in a complex response network that is driven by the cellular, molecular and chemical biology of poly(ADP-ribose) (PAR). This PAR-dependent response network is crucial for a broad array of physiological and pathological responses and thus is a good target for chemical therapeutics for several diseases.     

Poly ADP-ribose polymerase: self-ADP-ribosylation, the stimulation by DNA, and the effects on nucleosome formation and stability.

A partially purified preparation of the enzyme poly ADP-ribose polymerase which controls the transfer of ADP-ribose from NAD has been investigated. Data presented here indicate that the enzyme ADP-ribosylates itself. The enzyme preparation can be stimulated by DNA and this stimulation is exclusively associated with an auxiliary protein which copurifies with the enzyme and which we refer to as endogenous acceptor protein. Exogenously added proteins such as histones H1, H2A, and H3, cholera toxin, and Escherichia coli DNA-dependent RNA polymerase can also act as acceptor proteins in addition to the DNA-associated labeling of the endogenous acceptor. We speculate that the self-ADP-ribosylation of enzyme and that of the endogenous acceptor may play a role in control of the extremely rapid turnover of cellular NAD. Additionally, we have used this enzyme to ADP-ribosylate histones and to determine the effect of such modification on in vitro nucleosome formation and stability. The enzyme mediated ADP-ribosylation of free histones prior to incorporation into nucleosomes affects both nucleosome formation and stability while such ADP-ribosylation of histones already incorporated into nucleosomes does not affect their stability. These observations suggest that the ADP-ribosylation of histones prior to their involvement in nucleosomes might be the site of the physiologically important ADP-ribose transfer.     

Dynamic relocation of poly(ADP-ribose) glycohydrolase isoforms during radiation-induced DNA damage

Poly(ADP-ribosyl)ation is a very early cellular response to DNA damage. Poly(ADP-ribose) (PAR) accumulation is transient since PAR is rapidly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). PARG may play a prominent role in DNA damage response and repair by removing PAR from modified proteins including PARP-1. Using living cells, we provide evidence that in response to DNA damage induced by gamma-irradiation the cytoplasmic 103 kDa PARG isoform translocates into the nucleus. We further observed that the nuclear GFP-hPARG110 enzyme relocalizes to the cytoplasm in response to DNA damage. Using different GFP-PARG fusion proteins specific for the nuclear and cytoplasmic forms, we demonstrate their dynamic distribution between cytoplasm and nucleoplasm and a high mobility of major PARG isoforms by fluorescence recovery after photobleaching (FRAP). The dynamic relocation of all PARG isoforms presented in this report reveals a novel biological mechanism by which PARG could be involved in DNA damage response.     

Poly(ADP-ribosyl)ation in regulation of chromatin structure and the DNA damage response

Poly(ADP-ribose) (PAR) is a post-translational modification of proteins and is synthesised by PAR polymerases (PARPs), which have long been associated with the coordination of the cellular response to DNA damage, amongst other processes. Binding of some PARPs such as PARP1 to broken DNA induces a substantial wave of PARylation, which results in significant re-structuring of the chromatin microenvironment through modification of chromatin-associated proteins and recruitment of chromatin-modifying proteins. Similarly, other DNA damage response proteins are recruited to the damaged sites via PAR-specific binding modules, and in this way, PAR mediates not only local chromatin architecture but also DNA repair. Here, we discuss the expanding role of PAR in the DNA damage response, with particular focus on chromatin regulation.     

Detection and quantification of poly-ADP-ribosylated cellular proteins of spleen and liver tissues of mice in vivo by slot and Western blot immunoprobing using polyclonal antibody against mouse ADP-ribose polymer

Poly-ADP-ribosylation (PAR) of cellular proteins has been shown to have decisive roles in diverse cellular functions including carcinogenesis. There are indications that metabolic level of poly-ADP-ribosylated cellular proteins might indicate carcinogenesis and, therefore, could be potentially used in cancer screening program. Keeping in mind the limitations of currently available assays of cellular PAR, a new assay is being reported that measures the metabolic level of poly-ADP-ribosylated cellular proteins. The ELISA based slot and Western blot immunoassay used polyclonal antibody against natural, heterogeneous ADP-ribose polymers. It could be successfully employed to qualitatively and quantitatively assay metabolic levels of poly-ADP-ribosylated proteins of spleen and liver tissues of normal mice or mice exposed to dimethylnitrosamine for up to 8 weeks; potentially PAR of cellular proteins could be assayed in any tissue or biopsy. Implications of the results in cancer screening program have been discussed. (Mol Cell Biochem 278: 213–221, 2005)     

Poly(ADP-ribosylated) histones in chromatin replication

Poly(ADP-ribosylation) of histones and several other nuclear proteins seem to participate in nuclear processes involving DNA strand breaks like repair, replication, or recombination. This is suggested from the fact that the enzyme poly(ADP-ribose) polymerase responsible for this modification is activated by DNA strand breaks produced in these nuclear processes. In this article I provide three lines of evidence supporting the idea that histone poly(ADP-ribosylation) is involved in chromatin replication. First, cellular lysates from rapidly dividing mouse or human cells in culture synthesize a significant number of oligo- in addition to mono(ADP-ribosylated) histones. Blocking the cells by treatment of cultures with 5 mM butyrate for 24 h or by serum or nutrient depletion results in the synthesis of only mono- but not of oligo(ADP-ribosylated) histones under the same conditions. Thus, the presence of oligo(ADP-ribosylated) histones is related to cell proliferation. Second, cellular lysates or nuclei isolated under mild conditions in the presence of spermine and spermidine and devoid of DNA strand breaks mainly synthesize mono(ADP-ribosylated) histones; introduction of a small number of cuts by DNase I or micrococcal nuclease results in a dramatic increase in the length of poly(ADP-ribose) attached to histones presumably by activation of poly(ADP-ribose) polymerase. Free ends of DNA that could stimulate poly(ADP-ribosylation) of histones are present at the replication fork. Third, putatively acetylated species of histone H4 are more frequently ADP-ribosylated than nonacetylated H4; the number of ADP-ribose groups on histone H4 was found to be equal or exceed by one the number of acetyl groups on this molecule. Since one recognized role of tetraacetylated H4 is its participation in the assembly of new nucleosomes, oligo(ADP-ribosylation) of H4 (and by extension of other histones) may function in new nucleosome formation. Based on these results I propose that poly(ADP-ribosylated) histones are employed for the assembly of histone complexes and their deposition on DNA during replication. Modified histones arise at the replication fork by activation of poly(ADP-ribose) polymerase by unligated Okazaki fragments.     

Endogenous ADP-ribosylation in skeletal muscle membranes

The characteristics of ADP-ribosyltransferase activity in skeletal muscle membranes have been studied. The membrane enzymes can ADP-ribosylate exogenous substrates such as guanylhydrazones, polyarginine, lysozyme, and histones. The properties of the enzyme are investigated by using diethylaminobenzylidineaminoguanidine as a model substrate. Incubation of the membranes with [32P]adenylate-labeled NAD results in the labeling of a number of cellular proteins. Magnesium ions, detergents, and diethylaminobenzylidineaminoguanidine stimulated the ADP-ribosylation of membrane proteins, whereas L-arginine methyl ester and arginine inhibited ADP-ribosylation. The labeling of specific proteins in the sarcoplasmic reticulum and glycogen pellet is influenced significantly by detergents, nucleotides, and thiols. The hydroxylamine sensitivity of the ADP-ribose linkage in the membrane proteins is similar to that reported for (ADP-ribose)-arginine linkage. Snake venom phosphodiesterase digestion of the ADP-ribosylated membranes produces 5'-AMP as the major acid-soluble digestion product. The results suggest that the primary mode of modification is mono(ADP-ribosyl)ation. The ADP-ribosyltransferase activity in the membrane preparations is not extracted under conditions used for solubilization of extrinsic proteins, suggesting that the activity is associated with some integral membrane protein.     

ADP-ribosylation of proteins in nuclei and mitochondria from tissues rats of various age exposed gamma-radiation

Constitutive and gamma-induced ADP-ribosylation of nuclei and mitochondrial proteins in 2- and 29-month-old rats was studied. ADP-ribosylation was determined by binding of [3H]-adenin with the proteins after incubation of cellular organells in reaction mixture supplemented with [adenin-2,8-3H]-NAD. It was detected that the level of total protein ADP-ribosylation in the nuclei is 4.5-6.2 times higher than in the mitochondria. By inhibition of poly(ADP-ribose) polymerase (PARP) with 3-aminobenzamidine and treatment of ADP-ribosylated proteins with phosphodiesterase I, it was demonstrated that about 90% of [3H]-adenin bound by proteins in the nuclei and 70% in the mitochondria was the result of PARP activity. The level of total ADP-ribosylation of nuclear and mitochondrial proteins in the tissues of old rats was reliably lower than in young animals. This reduction of ADP-ribosylation in old animals is the result of the lower activity of PARP, not of mono(ADP-ribosyl) transferase (MART). The level of ADP-ribosylation of proteins in the nuclei of brain and spleen cells of 2-month-old rats irradiated with of 5 and 10 Gy was by 49-109% higher than in the control. At the same doses of radiation, the level of ADP-ribosylation of nuclear proteins in brain and spleen of old rats increased only by 29-65% compared to the control. Unlike cell nuclei, the radiation-induced activation of ADP-ribosylation in mitochondria was less expressed: the level of ADP-ribosylation increased by 34-37% in young rats and by 11-27% in old animals. This increased binding of ADP-ribose residues by the proteins of nuclei and mitochondria from tissues of gamma-irradiated rats is exceptionally conditioned by activation of poly(ADP-ribosyl)ation because the level of mono(ADP-ribosyl)ation remains constant. The results of this study enable the suggestion that poly(ADP-ribosyl)ation also occurs in the mitochondria of brain and spleen cells of the gamma-irradiated rats, though less pronounced than in cell the cell nuclei of these tissues. Thus, one of the probable causes of the less efficient repair of radiation-induced DNA damage in old organisms is a decline of both constitutive and induced poly(ADP-ribosyl)ation of proteins in cell nucleus and mitochondria.     

Inhibition of the transport of citrate during ADP-ribosylation of inner membrane proteins of the mitochondria

The ability of rat liver submitochondrial particles to catalyze NAD+ hydrolysis with a transfer of ADP-ribose residues to protein membranes has been demonstrated ADP-ribosylation is directly dependent on NAD+ concentration upon saturation with 1 mM NAD+ and is inhibited by physiological compounds (e.g., ATP, 10 mM; nicotinamide, 10 mM); besides, it is an artificial acceptor of ADP-ribose, arginine methyl ester. It was found that ADP-ribose is accepted by inner mitochondrial membrane protein, whose molecular masses amount to 25-30 kDa. The fact that 5'-AMP is a product of ADP-ribose degradation by snake venom phosphodiesterase suggests that the inner membrane vesiculate proteins are modified by mono(ADP-ribose). Covalent modification of membrane proteins by ADP-ribose leads to citrate transport inhibition in inner membrane vesicles the [14C]citrate uptake is significantly decreased thereby. The ability of ADP-ribosylation inhibitors to restore the citrate transport rate is suggestive of a direct regulatory effect of NAD+-dependent ADP-ribosylation on the activity of citrate-translocating system of inner mitochondrial membranes.     

SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress

ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein–protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo. ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo. Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.     

ADP-ribosylation of chromosomal proteins and mouse mammary tumor virus gene expression. Glucocorticoids rapidly decrease endogenous ADP-ribosylation of nonhistone high mobility group 14 and 17 proteins

The relationship between endogenous ADP-ribosylation of chromosomal proteins and glucocorticoid-regulated mouse mammary tumor virus gene expression was investigated in cultured mouse mammary tumor cells. It was observed that glucocorticoids quickly decreased endogenous (ADP-ribose)n on the nonhistone high mobility group (HMG) 14 and 17 proteins. The half-time for this loss was 8 and 17 min, respectively, for the two proteins. (ADP-ribose)n on HMG 1 and 2 and on histone H1 was less susceptible to hydrolysis during glucocorticoid treatment. The rapid loss of (ADP-ribose)n from HMG 14 and 17 occurred in the same time frame as the induction of mouse mammary tumor virus RNA synthesis by glucocorticoids in these cells (Young, H. A., Shih, T. Y., Scolnick, E. M., and Parks, W. P. (1977) J. Virol. 21, 139-149). 3-Amino-benzamide, a specific inhibitor of (ADP-ribose)n synthetase, increased mouse mammary tumor virus RNA levels with an accompanying decrease in endogenous ADP-ribosylation of HMG 14 and 17. These results show that a decrease in endogenous ADP-ribosylation of HMG 14 and 17 is a consequence of glucocorticoid action and suggest that loss of (ADP-ribose)n from these proteins may be an important event in mouse mammary tumor virus gene expression.     

Effects of nucleotides on activity of a purified ADP-ribosyltransferase from turkey erythrocytes

A transferase purified from turkey erythrocytes catalyzed the NAD-dependent ADP-ribosylation of proteins in the supernatant, particulate, and detergent-solubilized fractions of bovine thymus as well as several purified proteins. Nucleoside triphosphates increased the rate of ADP-ribosylation of multiple soluble proteins from thymus and several purified proteins by about twofold. With lysozyme as substrate and 10 mm nucleotide, the order of effectiveness was ATP > ITP = GTP > CTP = UTP. Half-maximal stimulation of ADP-ribose incorporation into lysozyme was observed with 2.5 mm ATP. App(NH)p and inorganic tri- and tetrapolyphosphate were less effective than ATP; ADP, AMP, cAMP, and inorganic pyrophosphate were ineffective. Enhancement of transferase-catalyzed ADP-ribosylation by ATP was observed only at low (20–200 μm) NAD concentrations; with lysozyme as substrate, however, the effect of ATP was not due to prevention of NAD hydrolysis during the assay, nor was it due to an effect on ionic strength. The transferase catalyzed the ADP-ribosylation of several purified proteins and, depending on the protein substrate, ATP either increased, decreased, or did not alter the rate of ADP-ribosylation. It appears that ADP-ribosylation of cellular proteins by endogenous ADP-ribosyltransferases may be subject to regulation by nucleoside triphosphates.