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)

We have examined a variety of conditions for solubilizing and electrophoresing cell proteins in order to define optimum conditions for studying proteins modified by ADP-ribosylation. We have identified conditions in which proteins can be quantitatively extracted from cells in an undegraded form with the protein-ADPribose linkages intact. Effective measures include boiling cells briefly (4 min) in the presence of 2% SDS and 2 M urea at pH 6.8. Both SDS and urea were present in the 6-18% gradient polyacrylamide gel matrix used for electrophoresis. Under these conditions good resolution of proteins of a wide molecular-weight range is obtained. This system has been used to compare protein ADP-ribosylation in non-transformed and polyma virus-transformed baby hamster kidney (BHK) fibroblasts, since the latter cells have a greater NAD+ ADP-ribosyltransferase activity (measured in isolated nuclei and permeabilized cells). Addition of DNAase to permeabilized BHK cells over the range 10-150 micrograms led to a progressively greater activation of transferase compared with controls. When PyY cells were used, however, maximum activation was achieved with only 10 micrograms of DNAase, further additions producing a successively smaller activation relative to control cells without added nuclease. There were also differences between these cells in response to salt. Addition of NaCl (to about 0.3 M) to BHK cells resulted in various extents of transferase activation, whereas any addition of NaCl to the incubate of permeabilized PyY cells decreased transferase activity. These different enzyme activities between this transformed and non-transformed cell line are for the most part not reflected in the protein modification profiles seen on autoradiograms of acrylamide gels after electrophoresis 32P-labelled proteins. A variety of proteins are modified and their molecular weights depend on the NA concentration in the permeabilized cell incubation. At 0.5 microM NAD+ there were two major acceptors with Mr values of 14 kDa and 30 kDa, and at 100 microM NAD+, three major acceptors, with Mr values of 19 kDa. 45 kDa and greater than 170 kDa. NAD concentrations of between 1 microM and 100 microM had no further effect on protein ADP-ribosylation profiles, except for the protein(s) of Mr greater than 170 kDa, pointing to a critical difference around 0.5-1.0 microM substrate. In some experiments, however, a difference was observed in the intensity of radioactivity in two bands. This may represent two different proteins, or a single protein modified to different extents.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A gel-electrophoretic analysis of protein ADP-ribosylation in polyoma virus-transformed and non-transformed BHK-21/C13 fibroblasts. 300 86

Botulinum C2 toxin is a microbial toxin which possesses ADP-ribosyltransferase activity. In human platelet cytosol a 43-kDa protein was ADP-ribosylated by botulinum C2 toxin. Labelling of the 43-kDa protein using [32P]NAD as substrate was reduced by unlabelled NAD and nicotinamide. The label was removed by treatment with snake venom phosphodiesterase. Half-maximal and maximal ADP-ribosylation occurred at 0.1 microgram/ml and 3 micrograms/ml botulinum C2 toxin, respectively. The Km value of the ADP-ribosylation reaction for NAD was about 1 microM. The peptide map of the ADP-ribosylated 43-kDa protein was almost identical with platelet actin. The ADP-ribosylated 43-kDa substrate protein bound to and was eluted from immobilized DNase I in a manner similar to G-actin. Trypsin treatment of platelet cytosol decreased subsequent ADP-ribosylation of the 43-kDa protein without occurrence of smaller labelled polypeptides. Purified platelet actin was also ADP-ribosylated by botulinum C2 toxin with similar characteristics found with actin in platelet cytosol. Phalloidin decreased the ADP-ribosylation of actin in platelet cytosol and of isolated platelet actin. Half-maximal and maximal, about 90%, reduction of actin ADP-ribosylation was observed at 0.4 microM and 10 microM phalloidin, respectively. ADP-ribosylation of purified actin, induced by botulinum C2I toxin, abolished the formation of the typical microfilament network. The data indicate that platelet G-actin but not F-actin is a substrate of botulinum C2 toxin and that this covalent modification largely affects the functional properties of actin.
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PMID:ADP-ribosylation of platelet actin by botulinum C2 toxin. 309 31

The genes encoding the S1 and S2 subunits of pertussis toxin were expressed in Escherichia coli under lac operon transcription and translation control with pUC8 and pUC18 as the expression vectors. Various versions of the subunits were detected with anti-S1 or anti-S2 monoclonal antibodies. Recombinant S1, but not S2, subunit contained the enzymatic NAD-glycohydrolase and NAD:Gi ADP-ribosyltransferase activities. Both activities were also expressed by a truncated version of the S1 subunit in which the 48 carboxy-terminal amino acid residues, including a predicted Rossman structure and one of the two cysteines, had been deleted. The epitope for an anti-S2 monoclonal antibody was localized to the N-terminal 40-amino-acid region of the S2 subunit. Both the S1 and S2 subunits expressed in E. coli reacted with human hyperimmune serum. The full length and the truncated recombinant S1 subunit also reacted in Western blots with a neutralizing and protective monoclonal anti-S1 antibody. The different versions of S1 and S2 subunits expressed in E. coli are useful for mapping active sites, epitopes, and regions that interact with receptors or the other subunits in the holotoxin. These recombinant subunits will also facilitate the development of a safer, new-generation vaccine against whooping cough.
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PMID:Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli. 311 86

Choleragen (cholera toxin) activates adenylate cyclase by catalyzing ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide-binding protein. It was recently found (Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142) that a bovine brain membrane protein known as ADP-ribosylation factor or ARF, which enhances ADP-ribosylation of Gs alpha, also increases the GTP-dependent NAD:arginine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase, and auto-ADP-ribosylation activities of choleragen. We report here the purification and characterization of two soluble proteins from bovine brain that similarly enhance the Gs alpha-dependent and independent ADP-ribose transfer reactions catalyzed by toxin. Like membrane ARF, both soluble factors are 19-kDA proteins dependent on GTP or GTP analogues for activity. Maximal ARF effects were observed at a molar ratio of less than 2:1, ARF/toxin A subunit. Dimyristoyl phosphatidylcholine was necessary for optimal ADP-ribosylation of Gs alpha but inhibited auto-ADP-ribosylation of the choleragen A1 subunit and NAD:agmatine ADP-ribosyltransferase activity. It appears that the soluble factors directly activate choleragen in a GTP-dependent fashion. The relationships of the ARF proteins to the ras oncogene products and to the family of guanine nucleotide-binding regulatory proteins that includes Gs alpha remains to be determined.
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PMID:Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain. 312 77

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.
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PMID:Endogenous ADP-ribosylation in skeletal muscle membranes. 312 54

A novel ADP-ribosyltransferase C3 was purified to homogeneity from filtrates of certain strains of Clostridium botulinum type C by ammonium sulfate precipitation, gel filtration, ion-exchange chromatography and heat treatment. The molecular mass of botulinum ADP-ribosyltransferase C3 was found to be 25 kDa. In the presence of [32P]NAD but not with [carbonyl-14C]NAD, C3 labelled 21-24-kDa protein(s) in membranes of human platelets and other tissues. The Km value of the ADP-ribosylation reaction for NAD was about 2 microM. Labelling of the 21-24-kDa protein(s) by C3 was largely reduced by addition of nicotinamide. Snake venom phosphodiesterase cleaved the ADP-ribose attached to the 21-24-kDa protein(s) by C3 and released 5'AMP. C3 catalyzed hydrolysis of [carbonyl-14C]NAD and released [carbonyl-14C]nicotinamide. ADP-ribosylation of 21-24-kDa platelet membrane protein(s) was biphasically regulated by Mg2+, Mn2+ and Ca2+. In the absence of free divalent cations GTP, GTP[gamma S] and GDP but not GDP[beta S], GMP, ATP or ATP[gamma S] increased labelling by C3. In the presence of Mg2+, GTP[gamma S] was inhibitory. Guanine nucleotides prevented heat inactivation of the substrate protein(s) with the rank order GTP[gamma S] = GTP = GDP greater than GDP[beta S] greater than GMP much greater than ATP = GMP = ATP[gamma S]. The data support the view that the novel ADP-ribosyltransferase C3 modifies eukaryotic 21-24-kDa GTP-binding protein(s).
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PMID:Botulinum ADP-ribosyltransferase C3. Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes. 312 9

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.
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PMID:Eukaryotic mono(ADP-ribosyl)transferase that ADP-ribosylates GTP-binding regulatory Gi protein. 312 40

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.
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PMID:Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum. 314 11

We have studied ADP-ribosyltransferase activity in platelet cytosol and electropermeabilized platelets. Cytosolic activity causes ADP-ribosylation or of a 37 kDa protein that is activated by increasing the concentration of potassium phosphate. ADP-ribosylation is inhibited by thiol reagents, an effect partially reversed by cholera toxin. In electropermeabilized platelets incubated with [alpha-32P]NAD, the 37 kDa protein is also ADP-ribosylated as are other proteins and albumin. Under these conditions, ADP-ribosylation is partially inhibited by nicotinamide. This experimental design could be used to determine the effect of cell agonists on endogenous ADP-ribosylation of proteins.
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PMID:Endogenous ADP-ribosylation in human platelets. 314 71

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)
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PMID:Purification and characterization of ADP-ribosylarginine hydrolase from turkey erythrocytes. 317 79


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