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

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

The EJ-ras gene was placed under the transcriptional control of the steroid-inducible mouse mammary tumor virus promoter/enhancer and introduced into Rat-1 fibroblasts, yielding the 14C cell line. When these cells were exposed to dexamethasone in vitro, EJ-ras mRNA was induced 15- to 20-fold, the cells grew in agar, and, after injection of cells into syngenic Fischer 344 rats, they produced lethal fibrosarcomas. Inhibitors of poly(ADP ribose) polymerase, which prevent the activation of the purified enzyme by a synthetic octadeoxyribonucleotide duplex, inhibited both in vivo tumorigenicity and in vitro growth in soft agar. The enzyme inhibitor 1,2-benzopyrone, which was studied in detail, and other polymerase inhibitors had no effect on EJ-ras mRNA or p21 protein expression. Poly(ADP ribose) polymerase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, EC 2.4.2.30] was inhibited by the drug in both untreated and dexamethasone-treated cells both in vitro and in vivo to the same extent, but biological consequences of enzyme inhibition were manifest only when the cells were in the transformed tumorigenic state.
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PMID:Prevention of tumorigenesis of oncogene-transformed rat fibroblasts with DNA site inhibitors of poly(ADP ribose) polymerase. 310 26

In mammalian cells, NAD+ serves a dual role as a respiratory coenzyme and as a substrate for the posttranslational poly(ADP-ribose) modification of chromatin proteins, catalyzed by the nuclear enzyme poly(ADP-ribose) polymerase [NAD+ ADP-ribosyltransferase, EC 2.4.2.30]. Biological evidence strongly suggests that poly(ADP-ribosyl)ation modulates chromatin functions, although the precise molecular mechanisms involved have not yet been elucidated. Here we describe conditions for the rapid uptake of exogenously supplied NAD+ by living hepatocytes in primary monolayer culture. Raising the intracellular NAD+ concentration by 70% caused a 5-fold increase of chromatin-bound poly(ADP-ribose). We conclude that the constitutive level of posttranslational poly(ADP-ribose) modifications of chromatin proteins in mammalian cells is related to the availability of NAD+, which varies in different physiological and pathological states. We propose that poly-(ADP-ribose) may serve a hitherto unrecognized function by signaling altered metabolic conditions to the chromatin and thus modulate its functions in tune with changing metabolic states.
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PMID:Poly(ADP-ribose) may signal changing metabolic conditions to the chromatin of mammalian cells. 310 32

A 20-kilodalton adenosine nucleotide-binding protein (A-protein) extracted from rod outer segments is shown to catalyze the cholera toxin-mediated ADP-ribosylation of GTP-binding protein (G-protein) from the outer segment. Radiolabel from [adenylate-32P] NAD+ was associated specifically with both the alpha-subunit of G-protein and with A-protein in the presence of activated cholera toxin. In the absence of added A-protein, G-protein appears to undergo ADP-ribosylation at a slower rate. In the absence of G-protein, A-protein was found to be labeled following incubation with [adenylate-32P]NAD+ and cholera toxin. In the presence of G-protein, a light-dependent component of A-protein labeling was observed. A-protein is a labile component of rod outer segments and has an affinity for ADP. The findings suggest that A-protein may act as an ADP-ribosyltransferase in the cholera toxin-mediated ADP-ribosylation of G-protein.
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PMID:A-protein catalyzes the ADP-ribosylation of G-protein from cow rod outer segments. 311 91

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

NAD+:Protein ADP-ribosyltransferase (EC 2.4.2.30) (ADPRT) was purified from human placenta by affinity chromatography. With the purified enzyme specific antibodies were raised and partial amino acid sequences were determined. To one of the amino acid sequences corresponding oligonucleotides were synthesized. A sized HeLa lambda gt11 cDNA library was constructed and screened. Positive clones were characterized to be ADPRT specific by immuno- and hybridization techniques. Clone ADPRT-G8 reacted with affinity chromatographically purified specific antibodies and with two specific oligonucleotides. The DNA of this clone detected an mRNA of about 4 kb, sufficient in size to code for the ADPRT with an Mr of 116,000. Partial sequence analysis of this clone confirmed its identity by revealing sequences which code for peptides which were found in cyanogen bromide (CNBr) fragments of the purified enzyme. The ADPRT-G8 clone was characterized with respect to its restriction pattern. The cloned ADPRT cDNA now opens the possibility to investigate the role of this enzyme in control of cellular functions.
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PMID:Isolation of a cDNA clone for human NAD+: protein ADP-ribosyltransferase. 312 32

Covalent modification of proteins by ADP-ribosylation is a major mode of protein regulation in eukaryotic cells. ADP-ribosyltransferases have been characterized from mammals but little is known about these enzymes in lower vertebrates. We purified an ADP-ribosyltransferase (E.C. 2.4.2.30) from trout (Salmo trutta faris) by affinity chromatography and characterized it. The 11,700-fold purified activity shows a major protein band at a molecular mass of 75,000 kDa in a SDS-polyacrylamide gel. In situ reactivation of SDS gels showed the 75,000 kDa protein to be enzymatically active, and additional enzymatically active bands at molecular masses of 115,000, 90,000 and 87,000 kDa, respectively. The enzyme is capable of poly-ADP-ribosylation. It crossreacts with affinity purified antibodies raised against human poly(ADP-ribose)synthetase and, except for the temperature optimum, its properties strongly resemble the mammalian enzymes, indicating the conserved character of nuclear ADP-ribosyltransferases. The trout enzyme is DNA- and histone-dependent, has an optimal pH between 8 and 9 and an apparent Km for NAD+ of 24 microM. The temperature optimum is 10 degrees C compared with 25 degrees C for the human enzyme. Known ADP-ribosyltransferase inhibitors also inhibit the enzyme from trout.
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PMID:ADP-ribosyltransferase is highly conserved: purification and characterization of ADP-ribosyltransferase from a fish and its comparison with the human enzyme. 312 83

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


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