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 substrate for ADP-ribosyltransferase from Clostridium botulinum was purified from the cytosol of bovine adrenal gland. Purification procedures consisted of ammonium sulfate fractionation, chromatographies on columns of DEAE-Sepharose and phenyl-Sepharose, gel filtration on a TSK-gel G3000SW column, and Mono Q fast protein liquid chromatography. On DEAE-Sepharose chromatography, the substrate activity was eluted in two separate peaks, and electrophoretic analyses revealed that the substrates in the two peaks are of similar molecular weight but different isoelectric points. The major peak of the substrate was further purified. It was purified about 1,800-fold with a recovery of 2.2% by the above procedures. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation showed a single protein band at Mr 22,000. The purified protein served as a substrate for botulinum ADP-ribosyltransferase and was maximally ADP-ribosylated to the extent of about 0.7 mol of ADP-ribose/mol of protein. A guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was co-purified with the ADP-ribosylation substrate, and the purified protein maximally bound about 0.5 mol of GTP gamma S/mol. GTP gamma S binding was effectively competed by GTP and GDP but not by GMP, ATP, and ADP. Thus, the ADP-ribosylation substrate is a GTP-binding protein. This protein, designated Gb (b for botulinum), is widely distributed in various tissues. It was rich in brain, pituitary, and adrenal glands, and poor in heart, smooth, and skeletal muscles.
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PMID:Purification and properties of the cytosolic substrate for botulinum ADP-ribosyltransferase. Identification as an Mr 22,000 guanine nucleotide-binding protein. 313 28

ADP-ribosylation of histones and non-histone nuclear proteins was studied in isolated nuclei during the naturally synchronous cell cycle of Physarum polycephalum. Aside from ADP-ribosyltransferase (ADPRT) itself, histones and high mobility group-like proteins are the main acceptors for ADP-ribose. The majority of these ADP-ribose residues is NH2OH-labile. ADP-ribosylation of the nuclear proteins is periodic during the cell cycle with maximum incorporation in early to mid G2-phase. In activity gels two enzyme forms with Mr of 115,000 and 75,000 can be identified. Both enzyme forms are present at a constant ratio of 3:1 during the cell cycle. The higher molecular mass form cannot be converted in vitro to the low molecular mass form, excluding an artificial degradation during isolation of nuclei. The ADPRT forms were purified and separated by h.p.l.c. The low molecular mass form is inhibited by different ADPRT inhibitors to a stronger extent and is the main acceptor for auto-ADP-ribosylation. The high molecular mass form is only moderately auto-ADP-ribosylated.
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PMID:ADP-ribosylation in isolated nuclei of Physarum polycephalum. 314 Jul 89

We attempted to characterize ADP-ribose-amino acid bonds formed by various bacterial toxins. The ADP-ribose-arginine bond formed by botulinum C2 toxin in actin was cleaved with a half-life of about 2 h by treatment with hydroxylamine (0.5 M). In contrast, the ADP-ribose-cysteine bond formed by pertussis toxin in transducin and the ADP-ribose-amino acid linkage formed by botulinum ADP-ribosyltransferase C3 in platelet cytosolic proteins were not affected by hydroxylamine. HgCl2 cleaved the ADP-ribose-amino acid bond formed by pertussis toxin in transducin but not those formed by botulinum C2 toxin or botulinum ADP-ribosyltransferase C3 in actin and platelet cytosolic proteins, respectively. NaOH (0.5 M) cleaved the ADP-ribose-amino acid bonds formed by botulinum C2 toxin and pertussis toxin but not the one formed by botulinum ADP-ribosyltransferase C3. The data indicate that the ADP-ribose bond formed by botulinum ADP-ribosyltransferase C3 differs from those formed by the known bacterial ADP-ribosylating toxins.
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PMID:Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferase C3 and pertussis toxin. 314 Aug 13

Benzamides are potent inhibitors of nuclear ADP-ribosyltransferase and have been extensively used to demonstrate the involvement of ADP-ribosylation in cellular function. When permeabilized L1210 cells are treated with 50 microM 3-acetylamidobenzamide (3-aab) the enzyme is inhibited. However, when 50 nM 3-aab is used a two-fold stimulation of enzyme activity is produced. This anomalous stimulation is obtained with benzamides and nicotinamides and is correlated with their activity as inhibitors. Strikingly the steady-state level of poly(ADP-ribose) in intact cells is increased by these low levels of inhibitors. The mechanisms of this effect and its consequences for the experimental use of benzamides are discussed.
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PMID:Benzamides can stimulate as well as inhibit the activity of nuclear ADP-ribosyltransferase. 314 Oct 76

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.
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PMID:3'-Deoxy-NAD+ as a substrate for poly(ADP-ribose)polymerase and the reaction mechanism of poly(ADP-ribose) elongation. 314 24

L-type pyruvate kinase (EC 2.7.1.40) purified from pig liver was ADP-ribosylated by incubation with NAD and ADP-ribosyltransferase purified from hen liver nuclei. Maximal incorporation of the ADP-ribose moiety from NAD into the L-type pyruvate kinase was 0.98 mol/mol of subunit. The Km values for NAD and L-type pyruvate kinase were 0.17 mM and 9.7 microM, respectively. ADP-ribosylation of the L-type pyruvate kinase resulted in suppression of the subsequent phosphorylation catalyzed by cAMP-dependent protein kinase. The ADP-ribosylation-induced suppression of phosphorylation of the L-type pyruvate kinase also resulted in suppression of the phosphorylation-induced inactivation. Amino acid analysis, after exhaustive sequential digestion of ADP-ribosyl-L-type pyruvate kinase with pepsin, aminopeptidase M and carboxy-peptidase B showed arginine to be the ADP-ribose-accepting amino acid. These results together with finding of the ADP-ribosyltransferase activity in mammalian liver cytosol (Moss, J. and Stanley, S.J. (1981) J. Biol. Chem. 256, 7830-7833) suggest that ADP-ribosylation may participate in the regulation of the L-type pyruvate kinase activity through changes in the rate of phosphorylation.
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PMID:ADP-ribosylation suppresses phosphorylation of the L-type pyruvate kinase. 334 9

A novel enzymatic activity, i.e., the catalysis of the formation of ADP-ribosylcysteine, was found in the cytosol of human erythrocytes. The NAD:cysteine ADP-ribosyltransferase was partially purified by sequential chromatographic steps on phenyl-Sepharose, phosphocellulose, and Sepharose CL-6B. The enzyme has an apparent molecular weight of 27,000 +/- 3,000, as determined by gel permeation. The formation of ADP-ribosylcysteine was associated with the stoichiometric release of nicotinamide from NAD. The enzyme was found to be highly specific toward cysteine and cysteine methyl ester as ADP-ribose acceptors. S-Benzoyl-L-cysteine, cystine, histidine, glutamic acid, arginine, arginine methyl ester, and agmatine were ineffective as acceptors for this enzyme.
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PMID:An NAD:cysteine ADP-ribosyltransferase is present in human erythrocytes. 359 54

Both the inhibitory and stimulatory guanine nucleotide-binding proteins of the adenylate cyclase complex were measured in erythrocyte membranes from patients with pseudohypoparathyroidism (PHP). The inhibitory guanine nucleotide-binding protein (Ni) of adenylate cyclase was measured by incorporation of [32P]ADP-ribose from [32P]NAD into the 39K subunit of Ni catalyzed by pertussis toxin. The ADP-ribosyltransferase activity of the toxin was expressed through incubation with dithiothreitol and erythrocyte membranes. Erythrocytes from 12 patients with PHP type I (PHP-I) had Ni values similar to those of 9 normal subjects and 2 patients with pseudopseudohypoparathyroidism. In 6 PHP-I patients, decreased activity of the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase, as determined by reconstitution of adenylate cyclase in the Ns-deficient membranes of cyc-S49 cells, corresponded with the reduced degree of ADP-ribosylation of the 42K subunit of Ns catalyzed by cholera toxin. These data suggest that the defect of Ns results in reduced stimulation of adenylate cyclase in some PHP-I patients, and that enhanced inhibition of the enzyme due to an increase in the 39K subunit of Ni does not account for the biochemical lesion in PHP-I patients.
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PMID:The stimulatory and inhibitory guanine nucleotide-binding proteins of adenylate cyclase in erythrocytes from patients with pseudohypoparathyroidism type I. 393 45

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.
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PMID:Kinetic mechanisms of two NAD:arginine ADP-ribosyltransferases: the soluble, salt-stimulated transferase from turkey erythrocytes and choleragen, a toxin from Vibrio cholerae. 393 59

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.
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PMID:Regulation of endogenously catalyzed ADP-ribosylation in adipocyte plasma membranes by Ca2+ and calmodulin. 393 99


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