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)

Two major forms of phospholipase D (PLD) activity, solubilized from rat brain membranes with Triton X-100, were separated by HPLC on a heparin-5PW column with buffer containing octyl glucoside. One form was completely dependent on sodium oleate for activity. The other, which was dramatically activated by the addition of ADP-ribosylation factor (ARF) 1 and guanine 5' [gamma-thio]triphosphate, required the presence of phosphatidylinositol 4,5-bisphosphate in the phosphatidylcholine substrate for demonstration of activity, as described by others. Oleate-dependent activity was unaffected by guanine 5' [gamma-thio]triphosphate, or phosphatidylinositol 4,5-bisphosphate. Both sodium oleate-and ARF-dependent activities catalyzed transphosphatidylation, thus identifying them as PLDs. ARF-dependent PLD was activated by recombinant ARF5 (class II) and ARF6 (class III), as well as ARF1 (class I). Myristoylated recombinant ARFs were more effective than their nonmyristoylated counterparts. ARFs were originally identified as activators of cholera toxin ADP-ribosyltransferase activity. The effects of recombinant ARF proteins from the three classes on cholera toxin activity (assayed under conditions identical to those used to assay PLD activity) did not, however, correlate with those on PLD, consistent with the notion that different aspects of ARF structure are involved in the two functions.
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PMID:Activation of rat brain phospholipase D by ADP-ribosylation factors 1,5, and 6: separation of ADP-ribosylation factor-dependent and oleate-dependent enzymes. 797 29

Poly(ADP-ribosyl)ation metabolism, a post-translational modification, involves two nuclear enzymes. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are responsible for the anabolism and catabolism of poly(ADP-ribose) polymer, respectively. PARG, despite being less abundant than PARP, is a crucial determinant of polymer metabolism which is known to be implicated in DNA repair and other cellular processes. Here, we describe modifications to improve the purification of PARG from calf thymus, in terms of both quantity and quality, which would allow biochemical and immunological studies. We also developed a zymogram to identify functional polypeptides exhibiting PARG activity. Purified and crude enzyme preparations from calf thymus were electrophoresed in two-dimensional gels. Samples were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing the polymer substrate in the form of automodified PARP after a nonequilibrium pH gradient electrophoresis. After renaturation of PARG in the gel, four isoforms of activity were clearly detected in the purified enzyme preparation. Even in the crude extract of the tissue, we could observe the major isoform of PARG. This technique will permit a better understanding of poly(ADP-ribose) catabolism and better characterization of PARG isoforms.
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PMID:Purification of poly(ADP-ribose) glycohydrolase and detection of its isoforms by a zymogram following one- or two-dimensional electrophoresis. 807 79

An arginine-specific mono-ADP-ribosyltransferase is expressed on the surface of differentiated mouse skeletal muscle cells and is anchored in the membrane via a glycosylphosphatidylinositol tail. Following incubation of intact cells with [adenylate-32P]NAD and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 97-kDa [32P]ADP-ribosylated protein was observed under reducing conditions and a 140-kDa complex under nonreducing conditions. The ADP-ribosylated protein was purified on a laminin affinity column. Based on its N-terminal sequence (FNLDVM-GAIRKEGEPGSLFGF) and a partial internal sequence (GLMRSEELSFVAGAP), the modified protein was identified as integrin alpha 7. Following partial trypsin digestion, a 39-kDa/79-kDa radiolabeled fragment was produced (reduced/nonreduced SDS-PAGE), narrowing the ADP-ribosylation site to a 39-kDa segment in the extracellular domain of integrin alpha 7. Labeling under optimal conditions was at least 0.4 mol of ADP-ribose/mol of integrin alpha 7. Selective expression of both ADP-ribosyltransferase and integrin alpha 7 in cardiac and skeletal muscle, a similar developmental appearance, and the apparently specific ADP-ribosylation, are consistent with a regulatory association between these proteins. ADP-ribosylation may modulate integrin receptor signaling and could play a significant role in the regulation of muscle cell function by the extracellular matrix.
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PMID:Integrin alpha 7 as substrate for a glycosylphosphatidylinositol-anchored ADP-ribosyltransferase on the surface of skeletal muscle cells. 824 57

Auto-ADP-ribosylation of arginine-specific ADP-ribosyltransferase purified from chicken peripheral heterophils was investigated. When the purified ADP-ribosyltransferase was analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Coomassie brilliant blue staining, two protein bands corresponding to the molecular masses of 27.5 and 28.0 kDa were detected. Both proteins were auto-ADP-ribosylated when they were examined by zymographic in situ gel assay without exogenous acceptor for ADP-ribose transfer. The automodification was inhibited by the acceptor, arginine or agmatine, and an inhibitor of arginine-specific ADP-ribosyltransferase, novobiocin. The ADP-ribose-transferase linkage was labile in 0.5 M hydroxylamine (pH 7.5). The automodified transferase was not chased by a large excess of nonradioactive NAD and did not catalyze transfer of its ADP-ribose to p33, an endogenous substrate protein for the transferase in heterophils, therefore, that automodified transferase cannot serve as an intermediate in ADP-ribosylation of other proteins. Auto-ADP-ribosylated transferase showed higher activity than did the unmodified transferase in catalyzing ADP-ribosylation of the basic acceptor such as poly(L-arginine) and p33 while to ADP-ribosylate the acidic proteins such as casein, the modified transferase was less active. Automodification of the transferase decreased polyanion-induced ADP-ribosylation of p33. Automodification of arginine-specific ADP-ribosyltransferase apparently alters the specificity of its own substrate.
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PMID:Automodification of arginine-specific ADP-ribosyltransferase purified from chicken peripheral heterophils and alteration of the transferase activity. 831 68

Exoenzyme S was purified > 1,500-fold from the culture supernatant fluid of Pseudomonas aeruginosa 388 at high yield without utilization of solvents or detergents. Two proteins, with apparent molecular sizes of 53 and 49 kDa, cofractionated with exoenzyme S activity. Rabbit anti-49-kDa-protein immunoglobulin G was prepared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified 49-kDa protein as immunogen. Anti-49-kDa-protein IgG inhibited the ADP-ribosyltransferase activity of purified exoenzyme S in a dose-dependent manner, which indicated a role for the 49-kDa protein in the ADP-ribosylation reaction. Analysis by ultrafiltration showed that exoenzyme S activity and the 53- and 49-kDa proteins cofractionated and that exoenzyme S was apparently > 300 kDa in size. Urea (8 M) and 1.0% Triton X-100 reversibly decreased the apparent molecular sizes of exoenzyme S activity and the 53- and 49-kDa proteins to between 30 and 100 kDa.
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PMID:Purification and characterization of exoenzyme S from Pseudomonas aeruginosa 388. 841 52

Previous studies in our laboratory have shown that nitric oxide (NO) gas enhances NMDA-stimulated release of preloaded tritiated norepinephrine ([3H]NA) from rat brain slices in a dose-dependent, oxygen-sensitive, and cyclic GMP-independent manner. In this study we have attempted to determine the mechanism for the enhancement of neurotransmitter release seen with NO. No-enhanced transmitter release was not due to buffer acidification or generation of NO degradation products, since reducing buffer pH below 7.3 inhibited NMDA-stimulated [3H]NA release and nitrite or nitrate ions (3-100 microM) had no significant effect on release. Carbon monoxide (CO, 10-300 microM), another diatomic gas with properties similar to NO including heme binding and guanylate cyclase activation, had no significant effect on depolarization-induced [3H]NA release. The NO effect was probably not due to mono-ADP-ribosylation of cellular proteins, since the ADP-ribosyltransferase (ADPRT) inhibitors nicotinamide (10 microM-10 microM) and luminol (1 microM-1mM) did not diminish the enhancement of transmitter release seen with NO. The NA reuptake inhibitor desmethylimipramine (DMI, 10 nM-10 microM) neither mimicked nor blocked the effect of NO, suggesting that NO was not acting via inhibition or reversal of the NA transporter. Similar to NO, the metabolic inhibitors sodium azide (NaN3, 0.1-3 mM), potassium cyanide (KCN, 0.1-3 mM), and 2,4-dinitrophenol (2,4-DNP, 10-300 microM) also dose-dependently enhanced NMDA-stimulated [3H]NA release. These results suggest that NO may enhance neurotransmitter release by inhibiting cellular respiration and perhaps ultimately via altering calcium homeostasis.
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PMID:Mechanism for nitric oxide's enhancement of NMDA-stimulated [3H]norepinephrine release from rat hippocampal slices. 853 39

Vibrio cholerae O1, No. 31, a strain isolated from a patient with mild diarrhea, produced mainly the unnicked cholera toxin. The amount of toxin that had accumulated in the cells was approximately 200 times lower than that secreted into the culture medium. When the unnicked toxin was purified by three successive column chromatographies and then extracted from the polyacrylamide gel, the unnicked toxin showed two bands corresponding to the A and B subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the A1 fragment was detected by trypsinization. Biological and enzymatic activities of the purified toxin with trypsinization were identical to those of cholera toxin from V. cholerae 569B as seen in the rabbit skin permeability test and the NAD:agmatine ADP-ribosyltransferase assay. DNA sequences of the A and B subunits were identical to those of the A- and B-subunit genes from the El Tor 2125 and classical 0395 strains, respectively. These data suggest that the wild V. cholerae strain, No. 31, produces a toxin identical to toxins previously reported in the literature and secretes it without accumulation in the cell, as is the case with other strains. However, strain No. 31's ability to nick the toxin is diminished compared with such abilities of other strains.
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PMID:A classical strain of Vibrio cholerae with diminished ability to process the proteolytically sensitive site in the A subunit of cholera toxin. 864 66

Recent evidence suggests that nitric oxide (NO) may function as a second messenger in the intracellular signal transduction pathways. We explored the possibility that NO was involved in the signal for triggering apoptosis in smooth muscle cells (SMCs). Chemical NO donors induced SMCs apoptosis in a concentration- and time-dependent manner. The membrane-permeable cGMP analogue, dibutyryl-cGMP, did not induce SMCs apoptosis, and the highly selective inhibitor of cGMP-dependent protein kinase, KT5823, was unable to inhibit the induction of NO-induced SMCs apoptosis. Inhibitor of ADP-ribosyltransferase slightly attenuated the induction of SMCs apoptosis by S-nitroso-N-acetyl penicillamine (SNAP). The inhibitor of Na+-H+ antiporter, amiloride, completely inhibited the induction of SMCs apoptosis by SNAP. These results demonstrate for the first time that NO can induce apoptosis in SMCs, suggesting that NO acts as a mediator in the development of atherosclerosis lesion via alterations in the number of SMCs. In addition, the results suggest that NO exert these effects through a pathway that does not involve guanylate cyclase and cGMP-dependent protein kinase.
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PMID:Nitric oxide donor SNAP induces apoptosis in smooth muscle cells through cGMP-independent mechanism. 866 Mar 29

To determine whether exotoxin A may affect the transport of fluid across the lung epithelium, two isogenic strains of Pseudomonas aeruginosa PA103 (10(8) colony-forming units), one (PA103 tox omega) with a structural gene mutation in exotoxin A, were instilled into the distal airspaces of anesthetized rats. PA103 parental strain, but not its mutant, stimulated the removal of fluid from the distal airspaces of the lung. Instillation of exotoxin A alone caused a dose-dependent increase in the fluid transport across the lung epithelium. Instillation of amiloride (10(-3) M) with exotoxin A demonstrated that this effect partially depended on increased uptake of sodium across the lung epithelium. The absence of stimulation after instillation of an exotoxin A mutant (PE delta Glu553) without ADP-ribosyltransferase activity demonstrated that the effect of exotoxin A depended on its ADP-ribosyltransferase activity. Finally, the instillation of exotoxin A in rats depleted of macrophages indicated that the effect of exotoxin A was not secondary to the activation of alveolar macrophages by this toxin. In conclusion, these results indicate that the in vivo release of exotoxin A by live airspace P. aeruginosa directly stimulates the fluid removal from the airspaces by the lung epithelium. This may alter the volume or composition of airway secretions, and may contribute to the lung disease in patients infected with P. aeruginosa.
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PMID:Exotoxin A stimulates fluid reabsorption from distal airspaces of lung in anesthetized rats. 877 92

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which catalyzes the transfer of ADP-ribose units from NAD+ to a variety of nuclear proteins under the stimulation of DNA strand break. To examine its role in DNA repair, we have been studying the interaction of PARP with other nuclear proteins using disulfide cross-linking, initiated by sodium tetrathionate (NaTT). Chinese Hamster Ovary (CHO) cells were extracted sequentially with Nonidet P40 (detergent), nucleases (DNase+RNase), and high salt (1.6 M NaCl) with and without the addition of a sulfhydryl reducing agent. The residual structures are referred to as the nuclear matrix, and are implicated in the organization of DNA repair and replication. Treatment of the cells with NaTT causes the crosslinking of PARP to the nuclear matrix. Activating PARP by pretreating the cells with H2O2 did not increase the cross-linking of PARP with the nuclear matrix, suggesting a lack of additional interaction of the enzyme with the nuclear matrix during DNA repair. Both NaTT and H2O2 induced crosslinks of PARP that were extractable with high salt. To shorten the procedure, these crosslinks were extracted from cells without nucleases and high salt treatment, using phosphate buffer. Using western blotting, these crosslinks appeared as a smear of high molecular weight species including a possible dimer of PARP at 230 kDa, which return to 116 kDa following reduction with beta-mercaptoethanol.
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PMID:Association of poly(ADP-ribose) polymerase with nuclear subfractions catalyzed with sodium tetrathionate and hydrogene peroxide crosslinks. 885 66


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