Cytoplasmic Poly(ADP-Ribose) Polymerase Associated with Free Messenger Ribonucleoprotein Particles in Rat Brain

Poly(ADP-ribose) polymerase associated with free cytoplasmic messenger ribonucleoprotein particles (free mRNP particles) carrying messenger RNA has been characterized in rat brain. There were first-order kinetics for NAD with an apparent Km for NAD of 90.5 +/- 0.70 microM and Vmax of 19.7 +/- 2.8 pmol ADP-ribose incorporated min-1 mg protein-1. Five poly(ADP-ribose) protein acceptors were identified in the Mr 37,000-120,000 range. It is hypothesized that ADP-ribosylation of specific free mRNP proteins might play a role in the derepression and translation of the silent mRNAs of free mRNP particles.     

Cell cycle variations in ADP-ribosylation of HeLa nuclear proteins

Changes in ADP-ribosylation of nuclear proteins during the HeLa cell cycle were determined. Portions of synchronized cultures were withdrawn at intervals and cells were permeabilized by resuspension in hypotonic buffer containing detergents. Nuclear proteins were radioactively labeled by incubating samples with [32P]NAD. Modified species were resolved using one-dimensional and two-dimensional polyacrylamide gel electrophoresis. Measurements of the incorporation of [32P]NAD by permeabilized cells showed that ADP-ribosylation is a significant modification throughout the cell cycle. A twofold increase was detected during S phase. Autoradiograms of one-dimensional sodium dodecyl sulfate-polyacrylamide gels revealed that many nuclear nonhistones are modified, though the major acceptors of 32P were the histones and a 116,000-Da species (poly(ADP-ribose) polymerase). The same modified proteins were present through the cell cycle, but densitometry of autoradiograms demonstrated a general increase in the level of incorporation in S phase. Autoradiograms of two-dimensional gels of nuclear proteins labeled with [32P]NAD were consistent with these results. Although nonhistones of isolated metaphase chromosomes show a substantial reduction in ADP-ribosylation, histone modification is essentially unchanged in metaphase.     

Increased protein ADPribosylation in HeLa cells exposed to the anti-cancer drug methotrexate

DNA single-strand breaks (about 200-300 per genome) were transiently detected during the first hour when HeLa cells were incubated for up to 24 h with 100 microM methotrexate. There was an expected increase in ADPribosyltransferase activity, which reached a maximum 2-3-fold stimulation at 3 h but which was still greater than in control cells after 24 h. When hypoxanthine (25 microM) was present in the incubations together with the methotrexate the transferase was no longer activated, although basal, control levels of activity were still present. DNA strand breaks were reduced in number but were still just detectable under these conditions. Cellular NAD+ levels were mostly unaffected by the various drug treatments, except for a small transient decrease after 1 h, possibly as a result of the transferase activation. Methotrexate did not cause an increase in the rate of ADPribose degradation. Degradation of ADPribose residues labelled in a preincubation period in permeabilized cells was more extensive at pH 6.0 was a 50% loss of acid-insoluble radioactivity in 30 min at 26 degrees C. At pH 8.0 the loss did not exceed 30-35% even after 90 min incubation. The activation of the transferase is reflected in a general increase in protein ADPribosylation detected by autoradiography of 32P-labelled proteins in 6.25-18.25%T gradient acrylamide gels. There were three major acceptors with molecular masses of 17, 100 and over 100 kDa, which could be respectively a histone, a transferase-derived peptide fragment and the transferase itself. When ADPribosyltransferase was inhibited with 3-amino-benzamide DNA single-strand breaks were no longer detected. However, this had no observably signficant effect on the kinetics of loss of cell viability (from Trypan blue uptake), cell number or colony-forming ability. Similar results are observed in most cases when the activation of the transferase, resulting from the incubation of cells with methotrexate, is inhibited by hypoxanthine. We conclude from such observations that the enhanced protein ADPribosylation seen in the cells exposed to methotrexate is a direct consequence of drug-exposure, but does not have any significant influence over the course of events leading ultimately to cell death.     

Diadenosine 5', 5"'-P1,P4-tetraphosphate stimulates processing of adp-ribosylated poly(ADP-ribose) polymerase

The effect of diadenosine 5', 5"'-P1,P4-tetraphosphate (Ap4A) on the time course and acceptors of poly(ADP-ribose) synthesis was studied in undamaged and N-methyl-N'-nitro-N-nitrosoguanidine-treated human lymphocytes. Analysis of protein acceptors of poly(ADP-ribose) revealed that treatment with Ap4A stimulated ADP-ribosylation of bands at molecular weights of 96,000, 79,000, and 62,000. Pulse-chase studies showed that these bands were produced as a result of an effect of Ap4A on the processing of ADP-ribosylated proteins rather than on the synthesis of newly ADP-ribosylated proteins. By incubating permeabilized cells in the absence or presence of Ap4A and purified poly(ADP-ribose) polymerase auto-ADP-ribosylated with [32P]NAD+, we showed that the Mr = 96,000, 79,000, and 62,000 bands were derivatives of the prelabeled enzyme. Our results indicate that normal human lymphocytes process auto-ADP-ribosylated poly(ADP-ribose) polymerase to specific lower molecular weight products and that this processing is stimulated by Ap4A.     

Guanidine group specific ADP-ribosyltransferase in murine cells

We have identified a guanidine group specific ADP-ribosyltransferase activity, capable of transferring an ADP-ribose group from NAD to a low molecular weight guanidine compound [p-(nitrobenzylidine)amino]guanidine and proteins such as histone and poly-L-arginine, in a variety of murine cell lines. The enzyme activity appears to be associated with an integral membrane protein of apparent molecular weight 30-33 kDa. Incubation of the viable cells in isotonic phosphate buffered saline with [32P]NAD results in the incorporation of label into cellular proteins. Dimethyl sulfoxide treatment of the cells downregulates the transferase activity as well as the ADP-ribosylation of cell proteins with extracellular NAD.     

Eukaryotic mono(ADP-ribosyl)transferase that ADP-ribosylates GTP-binding regulatory Gi protein

An NAD:cysteine ADP-ribosyltransferase designated ADP-ribosyltransferase C was purified approximately 35,000-fold from human erythrocytes with an 11% yield. The purified ADP-ribosyltransferase C exhibited one predominant protein band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight (Mr) of 28,500. The Km values for NAD and cysteine methyl ester were determined to be 65 and 4,400 microM, respectively. By using human erythrocyte inside-out membrane vesicles, the transferase C was found to ADP-ribosylate the alpha subunit (Mr = 41,000) of Gi, which is a substrate for pertussis toxin. The ADP-ribosylation of Gi alpha catalyzed by ADP-ribosyltransferase C was inhibited by pre-ADP-ribosylation with pertussis toxin. The linkage of ADP-ribose-Gi alpha in the membranes formed by ADP-ribosyltransferase C was as stable to hydroxylamine as that formed by pertussis toxin. These data represent the first demonstration that eukaryotic cells contain an ADP-ribosyltransferase which can catalyze the ADP-ribosylation of a cysteine residue in Gi alpha.     

Enzymic and nonenzymic mono ADP-ribosylation of proteins in skeletal muscle

The acceptors of endogenously catalyzed monoADP-ribosylation in the cell free extract from rat skeletal muscle was searched. The main acceptor proteins in particulate were electrophoretically 52, 80, 100, and greater than 200 kDa proteins in the presence of SDS, while that in cytosol were 36 and 39 kDa proteins. Although no ADP-ribosylation was observed in particulate when the substrate NAD+ was replaced by ADP-ribose, the same ADP-ribose adducts were also formed with higher degree in cytosol. These results indicate that an enzymic and nonenzymic monoADP-ribosylation occur separately in cytosol and particulate. One acceptor, 36 kDa protein, appears to be glyceraldehyde-3-phosphate dehydrogenase.     

Acceptors of poly(ADP-ribosylation) in differentiation inducer-treated and untreated Friend erythroleukemia cells

Acceptors of poly(ADP-ribosylation) were identified and compared between inducer-treated and untreated Friend erythroleukemia cells. When permeabilized Friend cells were pulse labeled with 0.6 μM [³²P]NAD for 1 min and labeled proteins analyzed by SDS-polyacrylamide gel electrophoresis, nucleosome core histones were found to be the primary acceptors, with an additional minor radioactive peak at a position corresponding to M_r = 170 000. Friend cells induced to differentiate by DMSO treatment showed a similar distribution of radioactivity, but with a 60% reduction in the overall level of poly(ADP-ribosylation) under identical labeling conditions. When isolated nuclei were pulse labeled with 0.6 μM [³²P]NAD, radioactive peaks were not restricted mainly at the positions of core histones but widely dispersed in the area from 10 to 50 kDa with another peak at 170 kDa. Increase of NAD concentration resulted in the overall shift of peaks to higher molecular weight positions. When pulse-labeled nuclei or permeable cells were chased with 1 mM NAD, radioactive peaks migrated to positions of very high molecular weight (>M_r = 180 000). Remarkable suppression of poly(ADP-ribose) synthesis was observed when DMSO, hexamethylene bisacetamide, butyric acid, or hemin were used as the inducers.     

Purification and characterization of ADP-ribosylarginine hydrolase from turkey erythrocytes

ADP-ribosylation of arginine appears to be a reversible modification of proteins with NAD: arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the opposing arms of the ADP-ribosylation cycle. ADP-ribosylarginine hydrolases have been purified extensively (greater than 90%) (150,000-250,000-fold) from the soluble fraction of turkey erythrocytes by DE-52, phenyl-Sepharose, hydroxylapatite, Ultrogel AcA 54, and Mono Q chromatography. Mobilities of the hydrolase on gel permeation columns and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions are consistent with an active monomeric species of approximately 39 kDa. Insertion of an organomercurial agarose chromatographic step prior to Ultrogel AcA 54 resulted in the isolation of a hydrolase exhibiting approximately 35-fold greater sensitivity to dithiothreitol (Ka,sensitive = 41 +/- 16.7 microM, n = 4; Ka,resistant = 1.44 +/- 0.12 mM, n = 3). A similar dithiothreitol-sensitive hydrolase was generated by exposure of the purified resistant enzyme to HgCl2. At 30 degrees C, both thiol-sensitive (HS) and thiol-resistant (HR) hydrolases were relatively resistant to N-ethylmaleimide (NEM); incubation with dithiothreitol prior to NEM resulted in complete inactivation. Both HS and HR required Mg2+ and thiol for enzymatic activity. Mg2+ stabilized both HS and HR against thermal inactivation in the absence and presence of thiol. A purified NAD:arginine ADP-ribosyltransferase, in the presence of NAD, inactivated both HS and HR; Mg2+ and to a greater extent Mg2+ plus dithiothreitol protected both HS and HR from NAD- and transferase-dependent inactivation. Thus, activation of the hydrolase enhanced its resistance to inactivation by transferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unexpected stimulation of mitochondrial ADP-ribosylation by cyanide

Cyanide, the classical inhibitor of the mitochondrial respiratory chain at site III, stimulates ADP-ribosylation of a number of mitochondrial proteins, the major protein being the 50-55 kDa band. Sodium azide, sharing the same inhibitory site, does not have the same effect. Rotenone or antimycin A have no influence on mitochondrial ADP-ribosylation. Data suggest that no apparent correlation exists between oxidoreductase function and protein ADP-ribosylation. Purified nuclear poly(ADP-ribose) polymerase activity was not affected by cyanide. The cyanide effect on mitochondrial ADP-ribosylation seems intriguing and may be attributed to NAD+-CN complex formation, since NAD reacts with cyanide at pH greater than 8 with N-substituted nicotinamide which may prevent inhibition of ADP-ribosylation.     

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50.A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA.Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.     

Demonstration and partial characterization of ADP-ribosylation in Pseudomonas maltophilia.

ADP-ribosylation of proteins occurs in many eukaryotes, and it is also the mechanism of action of a growing number of important bacterial toxins. To date, however, there is only one well-characterized ADP-ribosylation system where the ADP-ribosyltransferase and the substrate protein are both bacterial in origin, namely within the nitrogen-fixing bacterium Rhodospirillum rubrum. The present paper demonstrates the endogenous ADP-ribosylation of two proteins of Mr 32,000 and 20,000 within Pseudomonas maltophilia, a Gram-negative aerobe. The proteins have been partially purified: two apparently separate species of modified protein can be separated by ion-exchange chromatography and gel filtration (V0 and Mr 158,000 - Vi). The substrate protein(s) either has, or is co-eluted with, NAD+ glycohydrolase activity. The modification is mono-ADP-ribosyl in nature. The linkage between the acceptor amino acid and the ADP-ribose moiety is alkali-labile and stable to hydroxylamine, possibly indicating an S-glycosidic bond. The activity appears to be a true ADP-ribosylation reaction and not an NAD+ glycohydrolase activity followed by non-enzymic addition of ADP-ribose to protein. The results presented here indicate that ADP-ribosylation may have a wider significance within prokaryotic systems than previously thought.     

Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum

The enzyme that catalyzes the ADP-ribosylation and concomitant inactivation of dinitrogenase reductase in Rhodospirillum rubrum has been purified greater than 19,000-fold to near homogeneity. We propose dinitrogenase reductase ADP-ribosyltransferase (DRAT) as the working name for the enzyme. DRAT activity is stabilized by NaCl and ADP. The enzyme is a monomer with a molecular mass of 30 kDa and is a different polypeptide than dinitrogenase reductase activating glycohydrolase. NAD (Km = 2 mM), etheno-NAD, nicotinamide hypoxanthine dinucleotide, and nicotinamide guanine dinucleotide will serve as donor molecules in DRAT-catalyzed ADP-ribosylation reaction, and dinitrogenase reductases from R. rubrum, Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianium will serve as acceptors. No other proteins or small molecules, including water, have been found to be effective as acceptors. Nicotinamide is released stoichiometrically with formation of the ADP-ribosylated product. DRAT is inhibited by NaCl and has maximal activity at a pH of 7.0.     

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization.

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.     

CTP-dependent endogenous ADP-ribosylation of a 38 kDa protein in HL-60 cell membranes

Incubation of membranes of human promyelocytic leukemia HL-60 cells with [32P]NAD led to ADP-ribosylation of several proteins including a 38 kDa protein by endogenous ADP-ribosyltransferases. The ADP-ribosylation of the 38 kDa protein was distinctly different from others on the basis of pH dependency and heat stability at 50 degrees C, suggesting that there are at least two endogenous ADP-ribosyltransferases. It was enhanced by CTP, but not affected by ATP, GTP and UTP, whereas it was inhibited by GTP gamma S. [alpha-32P]CTP bound to the 38 kDa protein immobilized on a nitrocellulose sheet, indicating that the 38 kDa protein which bound CTP is strongly ADP-ribosylated by an endogenous ADP-ribosyltransferase.     

An extracellular monoADP-ribosyl transferase activity in Entamoeba histolytica trophozoites

Due to the important role of monoADP-ribosyl transferases in physiological and pathological events, we investigated whether the protozoan parasite Entamoeba histolytica had monoADP-ribosyl transferase activity. Reactions were initiated using ameba-free medium as the source of both enzyme and ADP-ribosylation substrate(s) and [32P]NAD+ as source of ADP-ribose. Proteins were analyzed by electrophoresis, and [32P]-labeled proteins were detected by autoradiography. Using the crude extracellular medium, a major labeled product of Mr 37.000 was observed. The yield of this product was reduced markedly using medium from Brefeldin A-treated trophozoites, indicating that the extracellular monoADP-ribosyl transferase and/or its substrate depended on vesicular transport. The labeling of the 37-kDa substrate was dependent on reaction time, temperature, pH, and the ratio of unlabeled NAD+ to [32P]NAD+. After two purification steps, several new substrates were observed, perhaps due to their enrichment. The reaction measured ADP-ribosylation since [14C-carbonyl]NAD+ was not incorporated into ameba substrates and a 75-fold molar excess of ADP-ribose caused no detectable inhibition of the monoADP-ribosyl transferase reaction. On the basis of sensitivity to NH2OH, the extracellular monoADP-ribosyl transferase of E. histolytica may be an arginine-specific enzyme. These results demonstrate the existence in E. histolytica of at least one extracellular monoADP-ribosyl transferase, whose localization depends upon a secretion process.     

Proteins ADP-ribosylated in Nuclei and Plasma Membrane Vesicles Isolated from Sea Urchin Embryos at Various Stages of Early Development

The ADP-ribosylations of proteins in nuclei, plasma membrane vesicles, mitochondria, microsome vesicles and the soluble fraction of sea urchin embryos isolated at various stages of development were examined by measuring the radioactivities of proteins after exposure of these subcellular fractions to [adenosine-¹⁴C]NAD or [adenylate-³²P]NAD. ADP-ribosylation of proteins was detected only in the nuclear and plasma membrane fractions. In the nuclear fraction, the rate of ADP-ribosylation of the histone fraction did not change appreciably during early development. In the TCA-insoluble protein fraction of the nuclei, the rate of ADP-ribosylation increased from fertilization to the morula stage, then decreased and again increased from the mesenchyme blastula to the late gastrula stage. After exposure of the nuclear fraction to [adenylate-³²P]NAD, a protein band with a molecular weight of 90 kDa was detected by SDS-polyacrylamide gel electrophoresis and radioautography at all stages examined. Its labeling intensity indicated that its ADP-ribosylation is higher at the morula and late gastrula stages than at other stages. In the plasma membrane fraction, proteins with molecular weights of 22 and 68 kDa were ADP-ribosylated and their rates of ADP-ribosylation hardly changed during early development.     

Kinetic mechanisms of two NAD:arginine ADP-ribosyltransferases: the soluble, salt-stimulated transferase from turkey erythrocytes and choleragen, a toxin from Vibrio cholerae

A subunit of choleragen and an erythrocyte ADP-ribosyltransferase catalyze the transfer of ADP-ribose from NAD to proteins and low molecular weight guanidino compounds such as arginine. These enzymes also catalyze the hydrolysis of NAD to nicotinamide and ADP-ribose. The kinetic mechanism for both transferases was investigated in the presence and absence of the product inhibitor nicotinamide by using agmatine as the acceptor molecule. To obtain accurate estimates of kinetic parameters, the transferase and glycohydrolase reactions were monitored simultaneously by using [adenine-2,8-3H]NAD and [carbonyl-14C]NAD as tracer compounds. Under optimal conditions for the transferase assay, NAD hydrolysis occurred at less than 5% of the Vmax for ADP-ribosylation; at subsaturating agmatine concentrations, the ratio of NAD hydrolysis to ADP-ribosylation was significantly higher. Binding of either NAD or agmatine resulted in a greater than 70% decrease in affinity for the second substrate. All data were consistent with a rapid equilibrium random sequential mechanism for both enzymes.     

Regulation of endogenously catalyzed ADP-ribosylation in adipocyte plasma membranes by Ca2+ and calmodulin

The effects of Ca2+ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [32P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca2+. Maximal incorporation of [32P] into the protein was achieved with free Ca2+ concentrations of 90 microM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 microM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [32P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca2+ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca2+ concentrations between 10(-6) - 10(-5) M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca2+ and calmodulin.     

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.