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)

ADP-ribosylation factors (ARFs) are approximately20-kDa guanine nucleotide-binding proteins that participate in vesicular transport in the Golgi and other intracellular compartments and stimulate cholera toxin ADP-ribosyltransferase activity. Both GTP binding and hydrolysis are necessary for its physiological functions, although purified mammalian ARF lacks detectable GTPase activity. An ARF GTPase-activating protein (GAP) was purified >15,000-fold from rat spleen cytosol using (NH4)2SO4 precipitation and chromatography on Ultrogel AcA 34, DEAE-Sephacel, heparin-Sepharose, hydroxylapatite, and Ultrogel AcA 44. In fractions ( approximately100-kDa proteins) from Ultrogel AcA 44, a major protein band of approximately50 kDa on SDS-polyacrylamide gel electrophoresis correlated with GAP activity, consistent with it being a homodimer, thus differing from an ARF GAP purified from rat liver (Makler, V., Cukierman, E., Rotman, M., Admon, A., and Cassel, D. (1995) J. Biol. Chem. 270, 5232-5237). Purified spleen GAP accelerated hydrolysis of GTP bound to recombinant ARF1, ARF3, ARF5, and ARF6; no effect of NH2-terminal myristoylation was observed. ARF GAP also activated GTP hydrolysis by ARL1, which is 56% identical in amino acid sequence to ARF1, but lacks ARF activity. ARD1 is a 64-kDa guanine nucleotide-binding protein that contains an 18-kDa ARF domain at its carboxyl terminus; the ARF domain lacks the amino-terminal alpha-helix found in native ARF and hence is similar to the amino-terminal truncated mutant Delta13ARF1. Both the ARF domain of ARD1 and Delta13ARF1 were poor substrates for ARF GAP. The non-ARF1 domain of ARD1 enhanced the GTPase activity of the ARF domain, but not that of the ARF proteins and Delta13ARF1, i.e. it lacks the relatively broad substrate specificity exhibited by ARF GAP.
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PMID:Characterization of a GTPase-activating protein that stimulates GTP hydrolysis by both ADP-ribosylation factor (ARF) and ARF-like proteins. Comparison to the ARD1 gap domain. 879 35

An arginine-specific ADP-ribosyltransferase activity was detected in chicken spleen membrane fraction using a capillary electrophoresis assay and the activity was extracted by phosphatidylinositol-specific phospholipase C but not by 1 M NaCl or 1% Triton X-100. The enzyme protein was purified from chicken spleen membrane fraction to apparent homogeneity with a six-step method containing phosphatidylinositol-specific phospholipase C treatment, ammonium sulfate precipitation and conventional column chromatographies. Apparent molecular mass of the purified enzyme estimated with SDS/PAGE was 44 kDa. N-glycanase treatment of the enzyme reduced the apparent molecular size on SDS/PAGE. The enzyme was recognized by anti-cross reacting determinant antibodies. Partial amino acid sequence of the purified enzyme protein showed high homologies with primary structures of previously reported chicken arginine-specific ADP-ribosyltransferases.
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PMID:A newly identified glycosylphosphatidylinositol-anchored arginine-specific ADP-ribosyltransferase in chicken spleen. 919 60

Chicken embryo cells were treated with caffeine (0.5-8.0 mM) alone or combined with various chemical and physical DNA-and/or chromatin-interactive agents. Analytical procedures comprised scheduled (SDS) and unscheduled (UDS) DNA synthesis, RNA synthesis (RNS), the activities of O6-alkylguanine-DNA alkyltransferase (AT) and poly (ADP-ribose) polymerase (PARP) as well as nucleoid sedimentation. Additional investigations were done in rat thymic and splenic cells. The effect of caffeine on DNase-I activity served as an in vitro-model system. When present in the PARP-, SDS-, UDS- and RNS-assays, caffeine inhibited the corresponding tracer (14C-NAD, dT-3H, 3H-U) incorporation in a dose-dependent manner. The AT activity was slightly stimulated. At concentrations of 0.06-0.3 mM, caffeine inhibited DNase-I activity by excess substrate. No specific effects of caffeine could be shown by nucleoid sedimentation. Besides the reduced permeability of the cells to nucleic acid precursors, the results obtained with the PARP- and DNase-I assays give evidence for the formation of a DNA-caffeine adduct as a prominent mechanism of cellular caffeine effects including DNA repair inhibition.
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PMID:Caffeine-DNA interactions: biochemical investigations comprising DNA-repair enzymes and nucleic acid synthesis. 930 78

RT6 is a rat lymphocyte glycosylphosphatidylinositol (GPI)-anchored alloantigen with nicotinamide adenine dinucleotide (NAD) glycohydrolase (NADase) and auto-ADP-ribosyltransferase activities. RT6 may have immunoregulatory properties based in part on the observation that injection of diabetes-resistant (DR)-BB rats with depleting doses of anti-RT6.1 mAb induced autoimmune diabetes and thyroiditis. We now report that injection of DR-BB rats with anti-RT6.1 mAb increased plasma NADase activity, which localized, by fluid phase liquid chromatography fractionation, to the high density lipoprotein (HDL) fraction. Following ultracentrifugation in high salt, however, RT6 was found in the nonlipoprotein fraction, where it existed, under nondenaturing conditions, as a 200-kDa complex and, by SDS-PAGE, as a 30- to 36-kDa species. Thy-1, another GPI-linked protein, and proteins that reacted with anti-GPI-oligosaccharide Abs also translocated from HDL to the nonlipoprotein fraction under similar conditions. Injection of anti-RT6.1 mAb into thymectomized DR and diabetes-prone-BB rats increased soluble RT6 to levels comparable to those observed in euthymic DR-BB rats, suggesting that HDL-bound RT6 is not derived from peripheral lymphocytes. In agreement, NADase activity in the plasma of eviscerated DR-BB rats did not increase following injection of anti-RT6 mAb. These data suggest that HDL is a carrier of plasma RT6 and other GPI-linked proteins, with equilibrium between the lipoprotein and nonlipoprotein fractions being salt dependent. Since GPI-linked proteins in HDL can transfer to cells in a functionally active form, the presence of RT6 in HDL is consistent with it having a role in signaling in nonlymphoid cells.
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PMID:Characterization of high density lipoprotein-bound and soluble RT6 released following administration of anti-RT6.1 monoclonal antibody. 968 81

CRM45 is a mutant form of diphtheria toxin (DTx) that lacks a 17-kDa carboxyl-terminal segment of the receptor-binding B subunit (DTB). The missing segment is a discrete structural domain of DTB that normally rests against the NAD binding pocket of the enzymically-active A subunit (DTA). Proteolytic cleavage and disulfide bridge reduction in the DTA-DTB linker region of DTx are required for optimal ADP-ribosylation of elongation factor 2 (EF-2). Here, we show that cleaved and uncleaved preparations of X-ray crystal grade CRM45 both exhibit an ADP-ribosyltransferase activity similar to that of cleaved DTx. Crystal-grade preparations of CRM45 also display a potent deoxyribonuclease activity. However, as observed with DTx, cleavage and reduction of CRM45 are not required for expression of this nuclease activity. After SDS-PAGE in a gel that contains DNA embedded in the matrix, renaturable Ca++/Mg(++)-dependent nuclease-active bands co-migrate with intact CRM45 (45 kDa) as well as with the DTA subunit (24 kDa) of CRM45. Because the 45-kDa nuclease-active band is unique to the CRM45 form of DTx, it offers direct proof that this activity is intrinsic to the DTA domain of DTx and its homologues.
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PMID:Characterization of the deoxyribonuclease and ADP-ribosyltransferase activities of CRM45, a truncated homologue of diphtheria toxin. 978 63

Here, we report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.
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PMID:Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase. 1019 6

T cells proteolytically shed the ectodomains of several cell surface proteins and, thereby, can alter their responsiveness and can release soluble intercellular regulators. ART2.2 is a GPI-anchored ecto-ADP-ribosyltransferase (ART) related to ADP-ribosylating bacterial toxins. ART2.2 is expressed exclusively by mature T cells. Here we show that ART2.2 is shed from the cell surface in enzymatically active form upon activation of T cells. Shedding of ART2.2 resembles that of L-selectin (CD62L) in dose response, kinetics of release, and sensitivity to the metalloprotease inhibitor Immunex Compound 3, suggesting that ART2.2, like CD62L, is cleaved by TNF-alpha-converting enzyme or by another metalloprotease. ART2.2 shed from activated T cells migrates slightly faster in SDS-PAGE analyses than does ART2.2 released upon cleavage of the GPI anchor. This indicates that shedding of ART2.2 is mediated by proteolytic cleavage close to its membrane anchor. Shed ART2.2 is enzymatically active and ADP-ribosylates several substrates in vitro. Thus, shedding of ART2.2 releases a potential intercellular regulator. Finally, using a new FACS assay for monitoring ADP-ribosylation of cell surface proteins, we demonstrate that shedding of ART2.2 correlates with a reduced sensitivity of T cell surface proteins to ADP-ribosylation. Our findings suggest that by shedding ART2.2 the activated T cell not only releases a potential intercellular regulator but also may alter its responsiveness to immune regulation by ART2.2-mediated ADP-ribosylation of cell surface proteins.
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PMID:Metalloprotease-mediated shedding of enzymatically active mouse ecto-ADP-ribosyltransferase ART2.2 upon T cell activation. 1103 85

Apoptosis (programmed cell death) is an active physiological mechanism from which removal of abundant or potentially harmful cells follows. Apoptosis of lymphocytes is critical for the development of the immune system and during the immune response. As we have shown previously, moderate osmotic cell shrinkage interferes with CD95(Fas/Apo-1)-induced cell death. The present study has been performed to further elucidate the underlying mechanisms. To this end, apoptosis in Jurkat T-lymphocytes was elicited by triggering the CD95-receptor with monoclonal CD95/Fas-antibody. Osmotic cell shrinkage which was induced by the addition of 100 mM NaCl, did not significantly interfere with CD95-induced phosphatidylserine exposure nor the activation of caspase 3 activity as determined by PARP cleavage, DEVD-AMC consumption, or the activation of PAK2-kinase. However, osmotic cell shrinkage almost abolished CD95-induced DNA fragmentation (as revealed by propidium iodide staining) and the activation of a DNase as evidenced from SDS-PAGE gel assay. Western blot analysis showed CD95-induced tyrosine phosphorylation of a nuclear protein of ca. 20 kD which comigrated with nuclease activity. This tyrosine phosphorylation was almost completely abolished by the addition of 100 mM NaCl. Furthermore, osmotic cell shrinkage blunted the CD95-induced activation of the Src-like kinase p56lck. It is concluded that different signaling pathways mediate FITC-Annexin-V binding and DNase activation. Only the latter is sensitive to osmotic cell shrinkage.
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PMID:Inhibition of CD95/Fas-induced DNA degradation by osmotic cell shrinkage. 1109 32

Clostridium perfringens type E iota toxin consists of two unlinked proteins designated as iota a (Ia; molecular mass approximately 47 kDa), an ADP-ribosyltransferase and iota b (Ib; molecular mass approximately 81 kDa) which binds to the cell surface and facilitates Ia entry into the cytosol. By Western-blot analysis, Ib incubated with Vero cells at 37 degrees C generated a cell-associated, SDS-insoluble oligomer of Ib (molecular mass>220 kDa) within 15 s, which was still evident 110 min after washing cells. Ib oligomerization was temperature, but not pH, dependent and was facilitated by a cell-surface protein(s). Within 5 min at 37 degrees C, cell-bound Ib generated Na(+)/K(+) permeable channels that were blocked by Ia. However, Ib-induced channels or oligomers were not formed at 4 degrees C. Two monoclonal antibodies raised against Ib that recognize unique, neutralizing epitopes within residues 632-655 either inhibited Ib binding to cells and/or oligomerization, unlike a non-neutralizing monoclonal antibody that binds within Ib residues 28-66. The Ib protoxin (molecular mass approximately 98 kDa), which does not facilitate iota cytotoxicity but binds to Vero cells, did not oligomerize or form ion-permeable channels on cells, and neither trypsin nor chymotrypsin treatment of cell-bound Ib protoxin induced large complex formation. The link between Ib oligomers and iota toxicity was also apparent with a resistant cell line (MRC-5), which bound to Ib with no evidence of oligomerization. Overall, these studies revealed that the biological activity of iota toxin is dependent on a long-lived, cell-associated Ib complex that rapidly forms ion-permeable channels in cell membranes. These results further reveal the similarities of C. perfringens iota toxin with other bacterial binary toxins produced by Bacillus anthracis and C. botulinum.
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PMID:Clostridium perfringens iota toxin: characterization of the cell-associated iota b complex. 1217 36

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.
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PMID:Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine. 1263 8


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