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 ras oncogene products (ras p21s) are 21-KDa proteins with activities of GTP binding and hydrolysis. A number of proteins homologous to ras p21 have been discovered and collectively named small molecular weight GTP-binding proteins. These proteins undergo post-translational modification with isoprenoid residues attached to cysteine in their carboxyl terminal. With this modification, they attach to cellular membranes. The biochemical activities of these proteins, i.e., GTP hydrolysis and binding, are regulated by various regulatory factors such as GDP-GTP exchange proteins and GTPase-activating proteins, but little is known about the cellular functions and physiological pathways through which they regulate these functions. Botulinum C3 ADP-ribosyltransferase, a 23-KDa exoenzyme secreted from certain strains of types C and D Clostridium botulinum, specifically ADP-ribosylates the rho family of these GTP-binding proteins. This ADP-ribosylation occurs at a specific asparagine residue in their putative effector domain, and presumably interferes with their interaction with a putative effector molecule downstream in signal transduction. C3 exoenzyme, when incubated with or microinjected into cultured cells, ADP-ribosylates a rho gene product in the cells, and causes profound cell rounding with loss of adhesion plaques and collapse of stress fiber. Microinjection of an activated mutant of rho A protein, on the contrary, induced extensive adhesion and actin assembly in cultured cells. These results suggest that the rho family of proteins are involved in morphogenesis and motility of cells via assembly and disassembly of cytoskeletal systems, and botulinum ADP-ribosyltransferase is a useful tool for clarifying the molecular mechanism of these processes.
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PMID:[ras oncogene-related small molecular weight GTP-binding protein, rho gene product and botulinum C3 ADP-ribosyltransferase]. 160 29

Pertussis toxin, a protein virulence factor produced by Bordetella pertussis, is composed of an A protomer and a B oligomer. The A protomer consists of a single polypeptide, termed the S1 subunit, which disrupts transmembrane signaling by ADP-ribosylating eukaryotic G-proteins. The B oligomer, containing five polypeptides, binds to cell receptors (most likely containing carbohydrate) and delivers the S1 subunit. Current knowledge suggests that expression of ADP-ribosyltransferase activity in target eukaryotic cells arises after 1) nucleotides and membrane lipids allosterically promote the release of the S1 subunit; and 2) the single disulfide bond in the S1 subunit is reduced by reductants such as glutathione. This model suggests conditions for the proper use of the toxin as an experimental reagent.
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PMID:Pertussis toxin and target eukaryotic cells: binding, entry, and activation. 161 92

The role of ADP ribosylation of proteins in the physiological regulation of sporulation in Streptomyces griseus was studied. We report here that both the activity of NAD+: arginine ADP-ribosyltransferase (ADPRT) and the pattern of ADP-ribosylated proteins showed characteristic changes during the life cycle in S. griseus 2682. Analysis off ADP-ribosylated proteins revealed that in a nonsporulating mutant of the parental wild-type (wt) strain (Bld7 mutant), both the activity of ADPRT and the pattern of ADP-ribosylated proteins were different from those of the parental strain. Addition of 3-aminobenzamide (3AB), the most potent inhibitor of ADPRT, inhibited sporulation of S. griseus 2682 and the A-factor (AF)-induced sporulation of S. griseus Bld7, but in both cases the inhibitory effect of 3AB was strictly age-dependent. Using [alpha-32P]GTP, we have demonstrated the presence of GTP-binding proteins in purified cell membranes of S. griseus 2682 and S. griseus Bld7. The same GTP-binding proteins were observed in Bld7 and the wt. AF stimulated the basal GTPase activity of cell membranes of S. griseus 2682 in a concentration-dependent manner, suggesting that GTP-binding proteins might be involved in the AF-induced sporulation process.
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PMID:The possible role of ADP ribosylation in physiological regulation of sporulation in Streptomyces griseus. 161 34

Bordetella pertussis produces a protein virulence factor termed pertussis toxin. Many candidate pertussis vaccines are based on the rationale that an immune response that neutralizes the virulence activities of this toxin, which are thought to arise from its catalytic ADP-ribosyltransferase activity, would be beneficial. The report describes two methods that quantify the inhibition of this activity by human serum. One, termed a direct assay, involves an initial incubation of toxin with serum, a second incubation that activates the toxin, and a third incubation that measures the ADP-ribosyltransferase activity of the mixture. The other assay, termed a plate assay, involves immobilization of the toxin, exposure of the immobilized toxin to serum and washing of the plate, and then activation and assay of the toxin's ADP-ribosyltransferase activity. The plate assay may be more selective than the direct assay in terms of identifying antibodies that neutralize the toxin in vivo. Sera from controls, selected patients presenting with cough, and vaccinated infants were first analyzed by the direct assay. In contrast to sera from controls, sera from several of the patients and vaccinated infants strongly inhibited activity. Dose-response curves of inhibition were determined for samples from three vaccinated infants by both the direct and plate assays. One of the samples had a dose-response curve of a different shape and thus differed not only in titer but also in functional characteristics. A comparison of inhibition of ADP-ribosyltransferase activity and neutralization in a CHO cell assay indicated that there was incomplete agreement between the two assays. Taken together, these results indicate that measurement of inhibition of ADP-ribosyltransferase activity by human serum is practical and may be useful in the evaluation of responses to pertussis vaccines.
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PMID:Detection of antibodies inhibiting the ADP-ribosyltransferase activity of pertussis toxin in human serum. 162 52

Deleting an important active-site residue of diphtheria toxin, glutamic acid-148, reduces the toxin's ADP-ribosyltransferase activity by a factor of greater than 10(4). We considered using this mutation to construct a recombinant toxoid for expression by live attenuated vaccines and explored second-site mutations that might cause reversion. Activity was partially restored by substituting glutamic acid for valine-147 or by extending the deletion by five residues toward the NH2 terminus, thereby placing glutamic acid-142 immediately adjacent to tyrosine-149. In both mutants the indicated glutamic acid may occupy a spatial locus similar to that of glutamic acid-148 in the unmutated protein. Simply deleting a crucial residue does not, therefore, provide confidence that a second-site mutation could not readily restore activity to a toxoid.
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PMID:Reversion of recombinant toxoids: mutations in diphtheria toxin that partially compensate for active-site deletions. 163 Nov 10

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho proteins in eukaryotic cells. Using the photoaffinity probe [alpha-32P]nicotinamide-2-azidoadenine dinucleotide, we have identified the adenine ring binding domain of the NAD+ binding site. The specificity of labeling was demonstrated by saturation effects and protection by the natural compound at physiologically relevant concentrations. Saturation of labeling was observed at 50 microM. Protection experiments indicated an 80% protection of labeling by 100 microM NAD+ when protein was photolyzed in the presence of 10 microM probe. Trypsin or Staphylococcus aureus V8 protease digestion of the photolabeled protein, along with boronate affinity chromatography and immobilized metal affinity chromatography, was used to specifically isolate the peptide region photolabeled with the probe. The peptide corresponded to Phe9-Gly19 near the N terminus.
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PMID:NAD+ binding site of Clostridium botulinum C3 ADP-ribosyltransferase. Identification of peptide in the adenine ring binding domain using 2-azido NAD. 163 27

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.
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PMID:Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins. 164 35

1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin. 2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity. 3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5'-O-(2-thiodiphosphate) or guanyl-5'-yl imidodiphosphate. 4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold. 5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc- mouse lymphoma membranes. 6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (alpha Gs). 7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of alpha Gs. 8. Endogenous ADP-ribosylation of alpha Gs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP. 9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.
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PMID:Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase. 164 18

Pertussis toxin (PT), an oligomeric exotoxin of Bordetella pertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid single-step procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (S1), 70% (S2), greater than 90% (S3), greater than 90% (S4), and 50% (S5), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes.
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PMID:Purification and immunological characterization of HPLC-purified pertussis toxin subunits. 165 40

Exoenzyme S is an ADP-ribosyltransferase enzyme distinct from exotoxin A that is synthesized and secreted by Pseudomonas aeruginosa. Yields of exoenzyme S are variable and depend on strain and growth conditions. Since certain medium additives are required for exoenzyme S production, its regulation may be influenced by environmental stimuli. In this study, we have cloned a region that complements the exoenzyme S-deficient phenotype of strain 388 exs1::Tn1, a chromosomal Tn1 insertional mutation. A large clone (28 kb) was shown to restore both synthesis and secretory functions to the mutant strain. Subcloning and Tn501 mutagenesis experiments localized the region required for exoenzyme S synthesis to a 3.2-kb fragment. Nucleotide sequence analysis demonstrated several open reading frames. Comparison of the N-terminal amino acid sequence of purified exoenzyme S with predicted amino acid sequences of all open reading frames indicated that the structural gene was not encoded within the sequenced region. Homology studies suggested that the region encoded three regulatory genes, exsC, exsB, and exsA. ExsA was homologous to the AraC family of transcriptional activator proteins, with extensive homology being found with one member of this family, VirF of Yersinia enterocolitica. VirF and ExsA both contain carboxy-terminal domains with the helix-turn-helix motif of DNA-binding proteins. The ExsA gene product appeared to be required for induction of exoenzyme S synthesis above a low basal level. Expression of ExsA was demonstrated by cloning the region under the control of the T7 promoter. Gene replacement experiments suggested that the expression of ExsC affects the final yield of exoenzyme S.
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PMID:Cloning and sequence analysis of a trans-regulatory locus required for exoenzyme S synthesis in Pseudomonas aeruginosa. 165 13


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