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:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Integrin alpha 7 is a major substrate in skeletal muscle cells for the cell surface, glycosylphosphatidylinositol-anchored, arginine-specific ADP-ribosyltransferase. Since ADP-ribosylarginine hydrolase, the enzyme responsible for cleavage of the ADP-ribosylarginine bond and a component with the transferase of a putative ADP-ribosylation cycle, is cytosolic, the processing of ADP-ribosylated integrin alpha 7 was investigated. Following incubation of differentiated mouse C2C12 myoblasts with [adenylate-32P]NAD and analysis by SDS-polyacrylamide gel electrophoresis under reducing conditions, two [32P]ADP-ribosylated forms of integrin alpha 7 were resolved. By pulse-chase and purification of the radiolabeled proteins on a laminin affinity column, it was demonstrated that a 105-kDa ADP-ribosylated form originated from a mono-ADP-ribosylated 102-kDa form and represented integrin alpha 7 modified at more than one site. The additional site(s) of modification, utilized at higher NAD concentrations, were located in the 63-kDa N-terminal segment of integrin alpha 7. Both [32P]ADP-ribosylated integrins were loosely associated with the cytoskeleton, bound to laminin affinity columns, and immunoprecipitated with antibodies to integrin beta 1. 32P label was rapidly removed from [32P]ADP-ribosylated integrin alpha 7 at either site of modification, a process inhibited by free ADP-ribose or p-nitrophenylthymidine-5'-monophosphate, an alternative substrate of 5'-nucleotide phosphodiesterase. The processed integrin alpha 7 was unavailable for subsequent ADP-ribosylation, although the amount of surface integrin alpha 7 remained constant. During the processing, no loss of label was observed from integrin alpha 7 radiolabeled with [14C]NAD, containing 14C in the nicotinamide proximal ribose, consistent with degradation of the ADP-ribose moiety by a cell surface 5'-nucleotide phosphodiesterase. Thus, cell surface ADP-ribosylation, in contrast to intracellular ADP-ribosylation, is not readily reversed by ADP-ribosylarginine hydrolase and seems to operate outside the postulated ADP-ribosylation cycle.
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PMID:Processing of ADP-ribosylated integrin alpha 7 in skeletal muscle myotubes. 772 41

This report demonstrates that incubation of cytotoxic T cells with NAD causes suppression of their ability to proliferate in response to stimulator cells or to lyse targets. Effects are evident after incubation for 3 h with concentrations of NAD as low as 1 microM and are sustained for many hours after removal of NAD from culture media. Suppression is a result of the failure of CTL to form specific conjugates with targets as well as a lower level of activation in response to TCR-mediated stimulation, although TCR-mediated transmembrane signaling is demonstrable. Metabolites of NAD such as nicotinamide, ADP-ribose, and cyclic-ADP-ribose have no detectable effect, indicating that NAD-glycohydrolase or ADP-ribose cyclase do not mediate suppression. Incubation of intact CTL with [32P]NAD leads to incorporation of 32P into a particulate, subcellular fraction, a reaction that is not inhibitable by ADP-ribose. Hydroxylamine, but not mercuric ion releases [32P]ADP-ribose, whereas phosphodiesterase releases [32P]AMP from the particulate subcellular fraction, suggesting that labeling is a result of enzymatic mono-ADP-ribosylation of arginines. In support of this, treatment of intact CTL with phosphatidylinositol-specific phospholipase C releases an arginine-specific ADP-ribosyltransferase and causes insensitivity to ecto-NAD suppression. These results suggest that a GPI-anchored ADP-ribosyltransferase uses ecto-NAD to ADP-ribosylate proteins that regulate CTL function.
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PMID:Regulation of cytotoxic T cells by ecto-nicotinamide adenine dinucleotide (NAD) correlates with cell surface GPI-anchored/arginine ADP-ribosyltransferase. 793 Jun 12

Nicotinamide and 3-aminobenzamide prevent TNF-alpha-mediated cytotoxicity, indicating that ADP-ribosylation plays a crucial role in this reaction. We have studied the role of ADP-ribosylation during TNF-alpha action in TNF-alpha-sensitive and TNF-alpha-resistant cells. Treatment of 3T3 cells with TNF-alpha, in the presence of [adenylate-32P]NAD followed by SDS-PAGE, revealed the involvement of specific ADP-ribosylation of a 90-kDa protein in TNF-alpha-mediated cytotoxicity. The stability of the ADP-ribosyl linkage on the 90 kDa protein in 100 mM 2-(N-cyclohexylamino)ethanesulfonic acid at pH 9.0 confirmed that ADP-ribosylation of the 90 kDa protein was mediated by an enzymatic reaction. Analysis of ADP-ribose residues by phosphodiesterase hydrolysis showed that the 90-kDa protein was modified by poly ADP-ribosylation. Poly ADP-ribosylation of the 90-kDa protein concomitant with cytotoxicity was observed in all TNF-alpha-sensitive but not TNF-alpha-resistant cells. Inhibition of ADP-ribosylation of the 90-kDa protein by benzamide but not by benzoic acid abrogated cytotoxicity, which further suggested that the poly-ADP-ribosylation of the 90-kDa protein is causally related to TNF-alpha-induced cell death. Our results demonstrate that TNF-alpha modifies a specific protein by poly-ADP-ribosylation during its action. Furthermore, ADP-ribosylation of specific proteins may be yet another mechanism regulating protein function during cellular metabolism.
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PMID:Poly ADP-ribosylation of a 90-kDa protein is involved in TNF-alpha-mediated cytotoxicity. 802 88

Denaturation of recombinant sarcosine oxidase or the natural enzyme isolated from Corynebacterium sp. P-1 with guanidine hydrochloride releases noncovalently bound FAD and a second UV-absorbing component (peak 2) which comigrates with NAD+ during reversed-phase HPLC. Both FAD and peak 2 are also found in extracts prepared by incubating sarcosine oxidase at 37 degrees C for 30 min, a procedure which causes partial (approximately 50%) release of the enzyme's noncovalently bound FAD. Peak 2 in the 37 degrees C extract is heat labile and decomposes upon boiling for 5 min at pH 8.0. A similar instability was observed with NAD+. Reaction of the 37 degrees C extract from sarcosine oxidase with phosphodiesterase yields nicotinamide mononucleotide, AMP, and FMN, as expected for a mixture containing NAD+ and FAD. Peak 2 was converted to NADH upon reaction of the 37 degrees C extract with yeast alcohol dehydrogenase in the presence of ethanol. Guanidine hydrochloride extracts, prepared from recombinant or natural enzyme, contain 1 mol of NAD+/mol of FAD. Since sarcosine oxidase contains 1 mol of noncovalently bound FAD, the results show that the enzyme also contains 1 mol of NAD+. The NAD+ is tightly bound and is not lost during enzyme purification. It is not susceptible toward hydrolysis by NADase, reduction by alcohol dehydrogenase, or nucleophilic attack by cyanide. Unlike the flavins in sarcosine oxidase, NAD+ is not reduced by sarcosine and is not in redox equilibrium with the flavins.
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PMID:Discovery of a third coenzyme in sarcosine oxidase. 852 44

The human leukocyte surface Ag CD38 was recently identified as a nicotinamide adenine dinucleotide (NAD)(+)-glycohydrolase ecto-enzyme, degrading NAD into nicotinamide and ADP-ribose. We show here that expression of CD38 is increased in the Jurkat T cell line after treatment with agents that augment intracellular cAMP, with the permeant cAMP analogue dibutyryl-cAMP (db-cAMP), and also with PMA, which activates protein kinase C. Treatment of human PBL T cells with db-cAMP or submitogenic doses of PMA also increased CD38 expression. Two other nucleotide-hydrolyzing activities were induced on the T cell surface concomitantly with CD38: the human PC-1 molecule, a nucleotide phosphodiesterase/pyrophosphatase that produces AMP from NAD or ADP-ribose, and a nucleotidase that produces adenosine from AMP, but which may be distinct from the CD73 5'-nucleotidase. All three enzymes were up-regulated after stimulation of human peripheral blood T cells with PHA. The coordinated regulation of these ecto-enzymes suggested that, besides a possible signaling function, they may recycle extracellular NAD by degrading it to adenosine and nicotinamide, which can be taken up by cells. In support of this hypothesis, db-cAMP-treated Jurkat cells could degrade extracellular NAD for de novo synthesis of purines, while untreated cells could not. Activated lymphocytes are often located in tissues in which cell death is common. It is suggested that the coordinated expression of these enzymes may allow activated T cells to re-use NAD and nucleotides from dead cells.
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PMID:Coordinated regulation in human T cells of nucleotide-hydrolyzing ecto-enzymatic activities, including CD38 and PC-1. Possible role in the recycling of nicotinamide adenine dinucleotide metabolites. 875 17

High levels of nitric oxide synthase and cyclic 3',5'-guanosine monophosphate (cGMP) in the olfactory bulb suggest that nitric oxide, acting as a diffusible intercellular messenger molecule inducing increased synthesis of cGMP, plays an important role in olfaction. The localization of cGMP after sodium nitroprusside stimulation of in vitro slices of rat olfactory bulb was compared with the distribution of nicotinamide adenine dinucleotide phosphatediaphorase, nitric oxide synthase, and glial fibrillary acidic protein. cGMP was detected immunohistochemically in cryostat sections. In the presence of the phosphodiesterase blocker isobutyl methylxanthine, cGMP was present in neurons in the glomerular layer, axons in the external and internal plexiform layers, and in a few somata and axons of the granule cell layer. This staining was blocked by NG-nitro-L-arginine methylester hydrochloride or hemoglobin. After sodium nitroprusside stimulation, the olfactory nerve layer was intensely stained, as were the glomeruli and periglomerular cells. In the external plexiform layer, axonal staining was increased substantially, and there were occasional multipolar cGMP-positive neurons. In the internal plexiform and granule cell layers, axonal staining was greatly increased. Many granule cells were also cGMP positive after sodium nitroprusside stimulation. cGMP and nitric oxide synthase-positive neuronal elements overlapped in the glomerular and granule cell layers, but staining was not colocalized, cGMP was not found in astrocytes. The glutamatergic antagonists D-2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline caused differential inhibition of cGMP accumulation in layers of the olfactory bulb. These findings support the hypothesis that nitric oxide is an intercellular messenger in the olfactory bulb (Breer and Shepherd [1993] Trends Neurosci. 16:5-9).
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PMID:Nitric oxide synthase, cGMP, and NO-mediated cGMP production in the olfactory bulb of the rat. 893 Jul 90

A membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified 215,000-fold from rabbit skeletal muscle and its gene was isolated from a skeletal muscle cDNA library. The enzyme was a glycosylphosphatidyl-inositol-linked protein, present on the surface of differentiated skeletal muscle myoblasts (myotubes). Following incubation of cultured, intact myotubes with [adenylate-32P]NAD and analysis by SDS-PAGE, a major radiolabeled protein of 97/140 kDa (reduced/nonreduced conditions) was observed. It was identified as integrin alpha 7 based on its size, binding to a laminin affinity column, immunoprecipitation with a monoclonal antibody, and partial amino acid sequencing. Since ADP-ribosylarginine hydrolase, the enzyme responsible for cleavage of the ADP-ribosylarginine bond and a component with the transferase of a putative ADP-ribosylation cycle, is cytosolic, whereas the transferase is attached via a GPI-anchor to the cell surface, the processing of ADP-ribosylated integrin alpha 7 was investigated. 32P label was rapidly removed from [32P]ADP-ribosylated integrin alpha 7, a process inhibited by free ADP-ribose or p-nitrophenylthymidine-5'-monophosphate, alternative substrates for 5'-nucleotide phosphodiesterase. The processed integrin alpha 7 was not susceptible to subsequent ADP-ribosylation, although the amount of surface integrin alpha 7 remained constant. During the processing, no loss of label was observed from integrin alpha 7 radiolabeled with [14C]NAD, containing 14C in the nicotinamide-proximal ribose, consistent with a degradation of the ADP-ribose moiety by a cell surface 5'-nucleotide phosphodiesterase. Thus, cell surface ADP-ribosylation, in contrast to intracellular ADP-ribosylation, is not readily reversed by the presently known ADP-ribosylarginine hydrolase and seems to operate outside the postulated ADP-ribosylation cycle.
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PMID:The alpha 7 integrin as a target protein for cell surface mono-ADP-ribosylation in muscle cells. 919 69

NAD may be degraded in several ways. A large number of investigations have shown that at least those catabolic routes which involve the formation of ADP-ribose are related to regulatory processes. In this study a rapid assay was utilized that permits identification of NAD-degrading enzymes directly in sodium dodecylsulfate polyacrylamide gels. Enzymatic activities were recovered by washing the gels in the presence of mild detergents such as lauryl dimethylamine N-oxide or Triton X-100. Subsequent incubation of the gels in the presence of the fluorescent analog 1,N6 etheno-NAD visualized NAD-degrading enzymes. Following excision of the fluorescent bands from the gels, the actual activity of the proteins was established by incubating the gel slices with 14C-labeled NAD and subsequent product analysis by thin layer chromatography (TLC). Homogenates from rat renal cortex and spleen were analyzed by this procedure. While in the spleen homogenate only a single band could be 'activity-stained', in the kidney three bands were detected. Kidney proteins with apparent molecular masses of about 210,000 and 105,000 Da were identified as phosphodiesterase and NAD pyrophosphatase (alkaline phosphodiesterase I), respectively. The third protein exhibited an apparent molecular mass of 41,000. The spleen protein (apparent molecular mass 45,000 Da) cleaved NAD to nicotinamide and ADP-ribose identifying it as NAD glycohydrolase. The procedure is suitable to screen for NAD-converting activities in crude extracts. It is specific for proteins which function as monomers or homo-oligomers.
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PMID:Detection and identification of NAD-catabolizing activities in rat tissue homogenates. 921 9

Previous immunohistochemical staining procedures of the brain and pituitary in Xenopus laevis, using an antiserum against neuronal nitric oxide (NO) synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, have revealed NOS activity in neurons and fibers in a number of brain areas, as well as in fibers in the pituitary. In the present study we have localized the target structures of the NOergic system in the Xenopus brain by visualizing the sites of NO-sensitive cyclic 3',5'-guanosine monophosphate (cGMP) accumulation, according to a method for cGMP visualization in rat brain slices. Brain slices of unfixed Xenopus are incubated in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine and the NO donor sodium nitroprusside, followed by fixation and cryosectioning. Sections were then processed for immunohistochemistry using rabbit and sheep antisera against cGMP and a sheep antiserum against nNOS. Visualization of single and double labeling of cGMP immunoreactive and/or nNOS immunoreactive structures was performed with combined CY3/fluorescein isothiocyanate fluorescence microscopy. Following this procedure, we provide immunohistochemical evidence for the distribution of cGMP-accumulating neurons in the brain of adult Xenopus. In most brain areas, the distribution of nNOS and cGMP immunoreactive structures (neuron somata and fibers) is distinct and separate, for instance in the dorsal pallium, the lateral thalamic nuclei, the optic tectum, the locus coeruleus and the reticular formation. However, nNOS and cGMP immunoreactive structures are often found in the vicinity of each other, and in the optic tectum even in adjacent neuron fibers and somata. The present observations are in line with the presence of an NO-dependent soluble guanylate cyclase in distinct brain areas of Xenopus laevis, corroborating similar data in the mammalian brain. Further, our observations may add to the understanding of the anatomical connectivity pattern and functional relevance of the NOergic system in the amphibian brain.
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PMID:Topographical relationship between neuronal nitric oxide synthase immunoreactivity and cyclic 3',5'-guanosine monophosphate accumulation in the brain of the adult Xenopus laevis. 971 Jan 48

1. Arginine-specific ADP-ribosyltransferase (ART) activity has been implicated in white cell chemotaxis. In this study, we examined the capacity of a panel of structurally unrelated inhibitors and pseudosubstrates of ART to inhibit chemotaxis of A7r5 rat vascular smooth muscle cells in response to PDGF-BB. 2. The IC50 values for nicotinamide (12 mM) and novobiocin (165 microM) were similar to those observed for inhibition of chemotaxis by human polymorphonuclear neutrophil leucocytes (PMN), whereas vitamins K3 (IC50=22 microM) and K1 (IC50=95 microM) were less potent than previously described in PMNs. The pseudo-substrates for the enzyme (DEA-BAG, agmatine and arginine-methylester) also inhibited A7r5 chemotaxis, and in addition inhibited cell adhesion at similar concentrations. Vitamin K3 was unique among the inhibitors of ART, in that it also inhibited cell adhesion. 3. A rat ART1 transcript was amplified by rtPCR from rat skeletal muscle, and was noted to share 94% homology with the mouse ART1 cDNA sequence. No such transcript could be detected in A7r5 cells by Northern blot analysis or rtPCR. 4. Evidence for ART activity on the surface of A7r5 cells was investigated using 32P-NAD+ as substrate, and labelled membrane proteins were observed with MWt values of 116, 100, 90 and 70 kDa. Exposure of the labelled proteins to phosphodiesterase yielded 32P-AMP, and hydrolysis with NaOH yielded 32P-NAD+. These results indicated that the labelled proteins were adducts with NAD+, and not the products of ART activity. The absence of ART catalytic activity in A7r5 cells was confirmed in protocols designed to show ADP-ribosylation of agmatine. 5. We conclude that the chemotactic activity of A7r5 cells is independent of ART activity, and the mechanism whereby the novel panel of inhibitors reduced cell migration remains undefined.
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PMID:Inhibition of chemotaxis in A7r5 rat smooth muscle cells by a novel panel of inhibitors. 977 55


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