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)

A mutant form of Pseudomonas aeruginosa exotoxin A (ETA) carrying a deletion of glutamic acid-553, an important active-site residue, was expressed in an ETA-negative strain of P. aeruginosa and shown to be exported from the cells as efficiently as wild-type ETA. The mutant protein, purified from the culture medium, was devoid of ADP-ribosyltransferase activity. Protein conformation was barely perturbed by the deletion, as determined by a number of measures, including affinity for substrate NAD, proteinase sensitivity, absorbance and fluorescence spectroscopy, and differential scanning calorimetry. The conformational integrity and stability of the mutant toxin are consistent with potential use of the protein in vaccines or as a carrier in preparing conjugate vaccines.
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PMID:Conformational integrity of a recombinant toxoid of Pseudomonas aeruginosa exotoxin A containing a deletion of glutamic acid-553. 134 36

Mono-ADP-ribosylation is a reversible modification of proteins, with NAD:arginine ADP-ribosyltransferases (EC 2.4.2.31) and ADP-ribosylarginine hydrolases (EC 3.2.2.19) catalyzing the opposing reactions in an ADP-ribosylation cycle. A membrane-associated arginine-specific (mono)-ADP-ribosyltransferase was purified 215,000-fold from rabbit skeletal muscle. On the basis of the amino acid sequences of HPLC-purified tryptic peptides, degenerate oligonucleotide primers were synthesized and used in a polymerase chain reaction (PCR)-based procedure to generate cDNA. A specific probe, based on PCR-generated sequence, was used to screen a rabbit skeletal muscle cDNA library. A composite cDNA sequence, obtained from library screening and rapid amplification of the 5' end of the cDNA, contained a 981-base-pair open reading frame, encoding a 36,134-Da protein. The deduced amino acid sequence contained the sequences of the tryptic peptides, hydrophobic amino and carboxyl termini, and two potential sites for N-linked glycosylation. Escherichia coli cells transformed with an expression vector containing transferase-specific sequence expressed ADP-ribosyltransferase activity. A transferase-specific oligonucleotide probe recognized a 4-kilobase mRNA expressed primarily in rabbit skeletal and cardiac muscle. There was no extended similarity in deduced amino acid sequences of the muscle transferase and several bacterial ADP-ribosylating toxins. The hydrophobic amino and carboxyl termini may represent a signal peptide and a site for a glycosyl-phosphatidylinositol anchor, respectively.
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PMID:Molecular characterization of NAD:arginine ADP-ribosyltransferase from rabbit skeletal muscle. 145 19

Poly(ADP-ribose)polymerase (PARP)-activity was assessed in vitro from the incorporation of the adenosine-diphosphate-ribose moiety of 14C-NAD+ in the acid-insoluble cell fraction. When compared to mammalian (rat) cells, chicken embryo cells exhibit an almost three- to fourfold higher constitutive PARP-activity and an about two- to threefold lower chromatin compactness as evidenced by viscometry of alkaline cell lysates and nucleoid sedimentation. X-irradiation, bleomycin and H2O2 activated PARP. Hyperthermia (43 degrees C), doxorubicin, ethidium bromide and novobiocin resulted in an inhibition of the enzyme activity. Even at the highest doses used, UV-light, monofunctionally alkylating agents and the bisbenzimide Hoechst 33258 remained without significant effects. It is suggested that, with respect to DNA-and/or chromatin-interactive agents, the chicken embryo PARP-test may be complementary to the results of morphological and biochemical studies.
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PMID:Poly(ADP-ribose)polymerase-activity of chicken embryo cells exposed to nucleotoxic agents. 146 59

Poly(ADP-ribosyl)ation is a eukaryotic posttranslational modification of proteins that is strongly induced by the presence of DNA strand breaks and plays a role in DNA repair and the recovery of cells from DNA damage. We compared poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) activities in Percoll gradient-purified, permeabilized mononuclear leukocytes from mammalian species of different maximal life span. Saturating concentrations of a double-stranded octameric oligonucleotide were applied to provide a direct and maximal stimulation of PARP. Our results on 132 individuals from 13 different species yield a strong positive correlation between PARP activity and life span (r = 0.84; P << 0.001), with human cells displaying approximately 5 times the activity of rat cells. Intraspecies comparisons with both rat and human cells from donors of all age groups revealed some decline of PARP activity with advancing age, but it was only weakly correlated. No significant polymer degradation was detectable under our assay conditions, ruling out any interference by poly(ADP-ribose) glycohydrolase activity. By Western blot analysis of mononuclear leukocytes from 11 species, using a crossreactive antiserum directed against the extremely well-conserved NAD-binding domain, no correlation between the amount of PARP protein and the species' life spans was found, suggesting a greater specific enzyme activity in longer-lived species. We propose that a higher poly(ADP-ribosyl)ation capacity in cells from long-lived species might contribute to the efficient maintenance of genome integrity and stability over their longer life span.
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PMID:Poly(ADP-ribose) polymerase activity in mononuclear leukocytes of 13 mammalian species correlates with species-specific life span. 146 94

6-Nitroso-1,2-benzopyrone and 3-nitrosobenzamide, two C-nitroso compounds that inactivate the eukaryotic nuclear protein poly(ADP-ribose) polymerase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, ADPRT, EC 2.4.2.30] at one zinc-finger site, completely suppressed the proliferation of leukemic and other malignant human cells and subsequently produced cell death. Tumoricidal concentrations of the drugs were relatively harmless to normal bone marrow progenitor cells and to superoxide formation by neutrophil granulocytes. The cellular mechanism elicited by the C-nitroso compounds consists of apoptosis due to DNA degradation by the nuclear calcium/magnesium-dependent endonuclease. This endonuclease is maintained in a latent form by poly(ADP-ribosyl)ation, but inactivation of ADPRT by C-nitroso drugs derepresses the DNA-degrading activity. ADPRT is thus identified as a critical regulatory enzyme component of a DNA-binding multiprotein system that plays a central function in defining DNA structures in the intact cell.
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PMID:Induction of endonuclease-mediated apoptosis in tumor cells by C-nitroso-substituted ligands of poly(ADP-ribose) polymerase. 150 87

Transducin is the retinal rod outer segment (ROS)-specific G protein coupling the photoexcited rhodopsin to cyclic GMP-phosphodiesterase. The alpha subunit of transducin is known to be ADP-ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP-ribosyltransferase activity. By using either ROS, cytosolic extract of ROS or purified transducin in the presence of [alpha-32P]nicotinamide adenine dinucleotide (NAD+), the alpha and beta subunits of transducin were found to be radiolabeled. The labeling was decreased by snake venom phosphodiesterase I (PDE I). The modification was shown to be mono ADP-ribosylation by analyses on thin layer chromatography of the PDE I-hydrolyzed products which revealed only 5'AMP residues. In addition we report that sodium nitroprusside activates the ADP-ribosylation of transducin.
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PMID:Mono ADP-ribosylation of transducin catalyzed by rod outer segment extract. 151 16

Mutants resistant to 3-aminobenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained from Streptomyces griseus IFO 13189, a streptomycin-producing strain. One (strain no. 4), which had significantly reduced ADP-ribosyltransferase activity, was analysed in detail. Mutant 4 displayed a conditional phenotype with respect to cultivation temperature. At 30 degrees C, it exhibited severely reduced ability to produce aerial mycelium (on solid medium) and submerged spores and streptomycin (in liquid culture), but this ability was fully restored at 25 degrees C. The mutant produced A-factor normally, regardless of cultivation temperature, and exhibited normal ability to accumulate ppGpp intracellularly. SDS-PAGE analyses of cellular proteins labelled by [32P]NAD revealed that an ADP-ribosylated protein with a molecular size of 44 kDa, which appeared in sporulating cultures of the parent strain, was missing from the mutant grown at the non-permissive temperature (30 degrees C). Genetic analysis showed that the aba mutation conferring resistance to 3-aminobenzamide was tightly linked to the altered phenotype. Failure to ADP-ribosylate certain cellular protein(s), presumably due to the aba mutation, may be responsible for impaired differentiation in this mutant.
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PMID:The possible role of ADP-ribosylation in sporulation and streptomycin production by Streptomyces griseus. 152 13

The poly ADP-ribosylation of proteins catalyzed by poly(ADP-ribose)polymerase (PARP) is involved in a number of important cellular metabolic activities. We evaluated various analogs of deoxythymidine and deoxyuridine as inhibitors of PARP. Most of these compounds have antiviral and/or anticancer activities. The structural requirements for these nucleoside analogs to be inhibitors of PARP were determined. The compounds evaluated had various substitutions on the 2-, 4- and/or 5-position of the pyrimidine ring, as well as on the 2'-, 3'- and/or 5'-position of the pentose moiety. Inhibition of PARP was strongly dependent on the size of the alkyl or halogen substituent on the 5-position of the pyrimidine ring. Whereas the 5-position of the pyrimidine ring could be varied, alteration of the 2- or 4-position drastically decreased the inhibition of PARP. Kinetic analysis was performed with concentrations of 1-10 microM NAD+. The Ki values for many compounds were five to seven times lower than the Ki for 3-aminobenzamide, a previously described potent inhibitor of PARP. Compounds with combined substituents at both the 5-position of the pyrimidine ring and the 3'- or 5'-position of deoxyribose generally were potent inhibitors of PARP, as for example 3'-amino-2', 3'-dideoxy-(E)-5-(2-bromovinyl)uridine (Ki = 0.7 microM), or 5'-azido-2',5'-dideoxy-5-ethyluridine (Ki = 0.8 microM). The 5-halogenated analogs had Ki values of 18, 35, 110 and greater than 1000 microM for 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine, respectively, and the 5-alkyl analogs had Ki values of 45, 2.2, 7, 16 and 180 microM for 5-methyl-2'-deoxyuridine, 5-ethyl-2'-deoxyuridine, 5-propyl-2'-deoxyuridine, 5-butyl-2'-deoxyuridine and 5-pentyl-2'-deoxyuridine, respectively. Two other compounds with substituents in the 5-position of the pyrimidine moiety also had potent activities: (E)-5-(2-bromovinyl)-2'-deoxyuridine (Ki = 6 microM) and 5-trifluoromethyl-2'-deoxyuridine (Ki = 1.6 microM). Compounds substituted in the 2'-, 3'- and/or 5'-position of the deoxyribose moiety were investigated and 5'-azido-5'-deoxythymidine, 5'-amino-5'-deoxythymidine, 3'-azido-3'-deoxythymidine and 3'-deoxythymidine (d2T) and Ki values of 12, 16, 18 and 30 microM, respectively.
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PMID:Inhibition of poly(ADP-ribose)polymerase activity by nucleoside analogs of thymidine. 153 Jun 62

Substitution of Tyr for His-426 of Pseudomonas aeruginosa exotoxin A results in a mutant protein with reduced ADP-ribosyltransferase activity (M. J. Wick and B. H. Iglewski, J. Bacteriol. 170:5385-5388, 1988). To investigate the role of His-426 in enzymatic activity, oligonucleotide-directed mutagenesis was used to construct mutant proteins encoding Ala, Glu, Gly, Lys, or Pro at position 426. The effect of these amino acid substitutions on ADP-ribosyltransferase activity was analyzed in 34,000-Da carboxy-terminal exotoxin A peptides (H426n peptides). ADP-ribosyltransferase activity of the H426n peptides fell within a range between 0.002 and 28% of wild-type levels of activity, suggesting that His-426 is required for full expression of enzymatic activity of exotoxin A. To investigate a possible catalytic function of His-426, the abilities of full-size (66,000-Da) wild-type exotoxin A and mutant proteins encoding either Ala-426 or Tyr-426 to hydrolyze NAD were compared by measuring NAD-glycohydrolase activity. This analysis revealed that exotoxin A encoding either Ala-426 or Tyr-426 expressed less than 1% of wild-type levels of NAD-glycohydrolase activity. Several criteria, including differential enzymatic activation properties and unique tryptic digestion patterns, revealed that the wild-type and mutant full-size proteins exhibit conformational differences. Our data suggest that His-426 plays a critical structural role in establishing the molecular architecture of the catalytic site in domain III and is important in orienting active-site residues in the cleft.
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PMID:Structure-function analysis of exotoxin A proteins with mutations at histidine 426. 154 28

Constitutive expression of human nuclear NAD+: protein ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose)polymerase; EC 2.4.2.30] as an active enzyme in Saccharomyces cerevisiae, under the control of the alcohol dehydrogenase promoter, was only possible with simultaneous inhibition of ADP-ribosylation by 3-methoxybenzamide. Induction of fully active pADPRT from the inducible galactose epimerase promoter resulted in inhibition of cell division and morphological changes reminiscent of cell cycle mutants. Expression of a pADPRT cDNA truncated at its 5' end had no influence on cell proliferation at all. Obviously the amino-terminal part of the DNA binding domain containing the first "zinc finger", which is essential for inducibility of pADPRT activity by DNA breaks, is also required for inhibition of cell growth on expression in yeast. Full-length as well as truncated pADPRT molecules were directed to the cell nucleus where the fully active enzyme produced large amounts of poly(ADP-ribose) by automodification. Since pADPRT turned out to be the only target for ADP-ribosylation in these cells, elevated levels of poly(ADP-ribose) were the most likely cause of inhibition of cell division, presumably resulting from interaction with chromosomal proteins.
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PMID:Inhibition of cell proliferation in Saccharomyces cerevisiae by expression of human NAD+ ADP-ribosyltransferase requires the DNA binding domain ("zinc fingers"). 155 29


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