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:3.5.4.4 (adenosine deaminase)
5,136 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Iodotubercidin is an adenosine kinase inhibitor that through its ability to increase levels of endogenous adenosine can enhance adenosine's receptor-mediated effects. We investigated whether iodotubercidin can inhibit [3H]adenosine accumulation by inhibiting transport processes in addition to inhibition of intracellular trapping of labeled adenine nucleotides. Under conditions in which extensive metabolism of intracellular adenosine was present, [3H]adenosine accumulation by DDT1 MF-2 cells was almost completely inhibited by iodotubercidin and the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine or by the nucleoside transport inhibitor nitrobenzylthioinosine. By using similar conditions, [3H]adenosine accumulation was significantly greater in Na+ buffer than in buffer containing N-methyl-D-glucamine in place of Na+; however, this effect may be explained by an observed 40% inhibition of adenosine kinase activity by N-methyl-D-glucamine. By using uptake intervals of 14 sec to represent the transport component of uptake, iodotubercidin decreased the affinity for adenosine, by about 3-fold, but had no effect on maximum velocity of transport. That these effects of iodotubercidin were due to direct interactions with nucleoside transporters was supported by findings that iodotubercidin inhibited [3H]nitrobenzylthioinosine binding to nucleoside transporters with a Ki value of 4 microM and inhibited [3H]uridine and [3H]formycin B uptake with IC50 values of 7 and 15 microM, respectively. These data suggest that iodotubercidin, at pharmacologically relevant concentrations, inhibits nucleoside transport independently of its well characterized inhibition of adenosine kinase and that N-methyl-D-glucamine must be used with caution in experiments to determine the possible presence of Na+ gradient-dependent concentrative nucleoside transporters.
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PMID:Effects of iodotubercidin on adenosine kinase activity and nucleoside transport in DDT1 MF-2 smooth muscle cells. 866 2

1. Previous studies have shown that the rat duodenum contains P1 and P2Y purinoceptors via which it relaxes to adenosine and adenosine 5'-triphosphate (ATP) respectively. It has also been shown to contract to uridine 5'-triphosphate (UTP) and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), and based on their differential inhibition by the P2 antagonist suramin it has been suggested that they act via two separate receptors. In addition, the rat duodenum has been shown to dephosphorylate ATP rapidly via ectonucleotidases and adenosine deaminase. In this study the responses of two preparations from the rat duodenum, the longitudinal muscle and the muscularis mucosae, were investigated using a series of nucleotides and suramin. 2. 2-Methylthioadenosine 5'-triphosphate (2-MeSATP), ATP, ATP-gamma-S and adenosine 5'-alpha,beta-methylene-triphosphonate (AMPCPP) each relaxed the longitudinal muscle, with an agonist potency order of 2-MeSATP > ATP = ATP-gamma-S > AMPCPP, while UTP and uridine 5'-diphosphate (UDP) were not observed to elicit relaxation. This indicates the presence of a relaxant P2Y-purinoceptor on the longitudinal muscle. The longitudinal muscle did not contract to any of the agonists at concentrations of 300 microM, apart from ATP-gamma-S which caused very weak contractions. 3. ATP-gamma-S, adenosine 5'-methylenediphosphonate (AMPCP), AMPCPP, ATP, UTP, adenosine 5'-diphosphate (ADP), UDP and 2-MeSATP each contracted the muscularis mucosae with an agonist potency order of ATP-gamma-S > or = AMPCP > or = AMPCPP = ATP = UTP = ADP = UDP >> 2-MeSATP, although maximal responses were not obtained at concentrations of 300 microM. The muscularis mucosae did not relax to any of the agonists at concentrations of 300 microM. 4. Suramin (1 mM) inhibited relaxations induced by ATP on the longitudinal muscle, shifting the relaxation concentration-response curve to the right. This further supports the presence of a P2Y-purinoceptor on this muscle layer. Suramin (1 mM) inhibited contractions induced by AMPCPP, but not those induced by ATP, UTP or ATP-gamma-S, in the muscularis mucosae. Desensitization of the muscularis mucosae was seen with AMPCPP, but not with UTP or ATP-gamma-S, and no cross-desensitization between AMPCPP and UTP or ATP-gamma-S was observed. This suggests there are two receptors which mediate contraction on the rat duodenum muscularis mucosae, one suramin-sensitive and the other suramin-insensitive. 5. ATP was rapidly degraded by the muscularis mucosae to ADP, adenosine 5'-monophosphate (AMP) and inosine, with no adenosine being detected. A similar rate of degradation was seen for UTP with UDP, uridine 5'-monophosphate (UMP) and uridine being formed and for 2-MeSATP with 2-methylthioadenosine 5'-diphosphate (2-MeSADP), 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) and 2-methylthioadenosine being formed. AMPCPP and ATP-gamma-S were both degraded more slowly, AMPCPP being degraded to AMPCP, and ATP-gamma-S to ADP, AMP and inosine. Suramin (1 mM), did not significantly affect the rate and pattern of degradation of these nucleotides, apart from AMPCPP which was degraded slightly more slowly in the presence of suramin. 6. These results show that there is a P2Y-purinoceptor which mediates relaxation in the rat duodenum longitudinal muscle. They also show that there is a contraction-mediating suramin-sensitive receptor on the rat duodenum muscularis mucosae which is desensitized by AMPCPP, and thus is probably of the P2X subtype. In addition, there is a contraction-mediating suramin-insensitive receptor on the rat duodenum muscularis mucosae which is not desensitized by UTP or ATP-gamma-S, and at which ATP and UTP show equal potency, and is thus probably of the P2U subtype. In addition, the rat duodenum muscularis mucosae contains ectonucleotidases and adenosine deaminase, which rapidly degrade nucleotides, although the inhibition by suramin of this deg
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PMID:Responses of the longitudinal muscle and the muscularis mucosae of the rat duodenum to adenine and uracil nucleotides. 885 97

We show here that 2'-deoxyadenosine (2'-dAdo) but not adenosine was toxic to chromaffin cells of 3-4-week-old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3-day period in the presence of 100 microM 2'-dAdo plus 3 microM deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase-mediated nick and labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis-like cell death by 2'-dAdo. Lethal effects of 2'-dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2'-dAdo-prompted cell death was not prevented by inhibitors of nucleoside transporter (3 microM dilazep or 1 microM nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 microM uridine or 100 microM 2'-deoxycytidine), or 5 mM nicotinamide. Cells incubated with 2'-dAdo (100 and 300 microM) showed a three- and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2'-dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2'-dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that this model could be used to study apoptosis and cell function.
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PMID:2'-deoxyadenosine induces apoptosis in rat chromaffin cells. 893 58

Extracellular adenosine triphosphate (ATP) has a range of effects on a wide variety of cells through the activation of specific purinoceptors. The aim of this study was to establish whether P2 purinoceptors are present on airway smooth muscle cells. Experiments were conducted on cultured rat tracheal smooth-muscle cells (first through third passage). Intracellular Ca2+ ([Ca2+]i) was measured using Fura-2 and dual-excitation wavelength microfluorometry. The effects of ATP, adenosine diphosphate (ADP), uridine triphosphate (UTP), and adenosine (ADO) were measured in concentrations from 10(-6) to 10(-3) M. At a concentration of 10(-4) M, the peak [Ca2+]i was 502 +/- 92 nM for ATP and 543 +/- 76 nM for UTP (mean +/- standard error of the mean). ADO had no significant effect on Ca2+ release. Peak [Ca2+]i induced by ATP was not dependent on extracellular Ca2+ but was blocked by U-73122, an inhibitor of phospholipase C. Pretreatment with adenosine deaminase and desensitization with alphabeta-MeATP had no effect on ATP-induced Ca2+ release. The effects of ATP (10(-4) M) on peak [Ca2+]i were potentiated by the presence of ADO 10(-5) M (969 +/- 257 nM; P < 0.05). The presence of XAC, a blocker of A1 and A2 ADO receptors did not prevent this effect. In the presence of XAC, ADO 10(-6) M potentiated the effects of ATP (peak [Ca2+]i: 1,300 +/- 229 nM). The addition of 1433U83, a blocker of A3 ADO receptors, blocked the synergistic effect of ADO 10(-6) M on ATP. These data show that P2 purinoceptors, most likely of the P2U subtype, are present on airway smooth muscle cells and that the newly discovered A3 ADO receptor appears to be also present.
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PMID:Effects of purine nucleotides and nucleoside on cytosolic calcium levels in rat tracheal smooth muscle cells. 903 28

8-Chloro-adenosine, the dephosphorylated metabolite of the antineoplastic agent 8-chloro-cyclic AMP, has been proposed to act on the regulatory subunits of cyclic AMP-dependent protein kinase. 8-Chloro-adenosine has a growth-inhibitory effect, the mechanism of which is unclear. We investigated the effects of 8-chloro-cyclic AMP and 8-chloro-adenosine on nucleic acid synthesis and cell cycle kinetics in two human glioma cell lines. These effects were compared to those of the cyclic AMP analogue 8-(4-chlorophenyl)-thio-cyclic AMP (8-CPTcAMP), which is less susceptible to dephosphorylation. Whereas 8-CPTcAMP almost completely inhibited RNA and DNA synthesis, both 8-chloro-adenosine and 8-chloro-cyclic AMP only partly inhibited synthesis of RNA and DNA at growth-inhibitory concentrations, as demonstrated by using [5-1H] uridine and [14C]thymidine incorporation. Therefore, the growth-inhibitory effect of 8-chloro-cyclic AMP is not (or not completely) due to activation of cyclic AMP-dependent protein kinase nor to the inhibition of nucleic acid synthesis. Flow cytometric analysis revealed that 8-chloro-cyclic AMP and 8-chloro-adenosine probably block cell cycle progression at the G2M phase. The effects of 8-chloro-cyclic AMP on nucleic acid synthesis and cell cycle progression were largely prevented by adenosine deaminase, which inactivates 8-chloro-adenosine. This indicates that the effects of 8-chloro-cyclic AMP were at least in part due to its metabolite 8-chloro-adenosine. Incorporation of 8-chloro-adenosine into RNA and DNA might contribute to the disturbance of the cell cycle kinetics and growth-inhibitory effect of 8-chloro-adenosine.
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PMID:The antiproliferative effect of 8-chloro-adenosine, an active metabolite of 8-chloro-cyclic adenosine monophosphate, and disturbances in nucleic acid synthesis and cell cycle kinetics. 903 46

Crystal structures of the cytidine deaminase-uridine product complex prepared either by cocrystallizing enzyme with uridine or by diffusing cytidine into ligand-free crystals show that the product binds as a 4-ketopyrimidine. They reveal four additional features of the catalytic process. (1) A water molecule bound to a site previously observed to bind the incoming 4-NH2 group represents the site for the leaving ammonia molecule. The conserved Pro 128 accommodates both moieties by orienting the carbonyl group of the previous residue. (2) The Glu 104 carboxylate group rotates from its hydrogen bond to the O4 hydroxyl group in transition-state analog complexes, forming a new hydrogen bond to the leaving group moiety. Thus, after stabilizing the hydroxyl group in the transition state, Glu 104 transfers a proton from that group to the leaving amino group, promoting enol-to-keto isomerization of the product. (3) Difference Fourier comparisons with transition-state complexes indicate that the pyrimidine ring rotates toward the zinc by approximately 10 degrees. The active site thus "pulls" the ring and 4-NH2 group in opposite directions during catalysis. To preserve coplanarity of the 4-keto group with the pyrimidine ring, the N1-C1' glycosidic bond bends by approximately 19 degrees out of the ring plane. This distortion may "spring-load" the product complex and promote dissociation. Failure to recognize a similar distortion could explain an earlier crystallographic interpretation of the adenosine deaminase-inosine complex [Wilson, D. K., & Quiocho, F. A. (1994) Nat. Struct. Biol. 1, 691-694]. (4) The Zn-Sgamma132 bond, which lengthens in transition-state complexes, shortens as the O4 atom returns to a state of lower negative charge in the planar product, consistent with our previous proposal that this bond buffers the zinc bond valence, compensating buildup of negative charge on the oxygen nucleophile during catalysis.
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PMID:The structure of the cytidine deaminase-product complex provides evidence for efficient proton transfer and ground-state destabilization. 912 97

Recently, we have demonstrated that adenosine and 2'-deoxyadenosine are toxic to embryonic sympathetic neurons and proposed that purine and pyrimidine metabolism may play a critical role in the growth and development of sympathetic neurons. To extend this hypothesis further, we examined the effects of these nucleosides on two other neuronal populations in the chick embryo, sensory dorsal root ganglion neurons and parasympathetic ciliary ganglion neurons. Now, we show that 2'-deoxyadenosine and adenosine have no visible adverse effect on the viability of either sensory or parasympathetic neurons. Instead, 2'-deoxyadenosine proved to be highly toxic to the nonneuronal cells. The toxic effects of 2'-deoxyadenosine were markedly enhanced by inhibition of adenosine deaminase. In contrast, adenosine was much less toxic to nonneuronal cells than 2'-deoxyadenosine and its effect was not potentiated by inhibition of adenosine deaminase. Priming of pyrimidine pools by exogenous uridine and the specific inhibitor of the nucleoside transporter, nitrobenzylthioinosine, did not protect nonneuronal cells from 2'-deoxyadenosine toxicity. Since phosphorylation of internalized nucleosides was a key step in the initiation of toxicity in sympathetic neurons, adenosine kinase activity was compared in sensory and sympathetic neuronal cultures. The adenosine kinase activity in dorsal root ganglion cultures was only 20% of that in sympathetic ganglion cultures. Furthermore, inhibition of phosphorylation by blocking 2'-deoxyadenosine kinase with iodotubercidin and 5'-amino-5'-deoxyadenosine had no protective effect against 2'-deoxyadenosine toxicity. [3H]-thymidine incorporation was inhibited over 90% by 2'-deoxyadenosine as early as 6 h following its addition and for up to 4 days, suggesting inhibition of proliferation of nonneuronal cells by 2'-deoxyadenosine. The nucleoside was also able to wipe out already well established nonneuronal cells, leaving behind an enriched population of sensory neurons. The selective vulnerability of nonneuronal cells to 2'-deoxyadenosine offers a convenient and effective tool for removing nonneuronal cells from neuronal cultures as well as providing a new model for studying the mechanisms of nucleoside toxicity.
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PMID:2'-Deoxyadenosine selectively kills nonneuronal cells without affecting survival and growth of chick dorsal root ganglion neurons. 955 58

This study was carried out to evaluate the possible role of adenosine uptake and metabolism in mediating the inhibitory actions of this nucleoside on spontaneous mouse oocyte maturation. Uridine blocked 3H-adenosine uptake by oocyte-cumulus cell complexes (OCCs) and cumulus cell-enclosed oocytes (CEOs) by 82-85%, whereas uptake by denuded oocytes (DOs) was suppressed by 97%. Uridine had no effect on germinal vesicle breakdown (GVB) in CEOs when meiotic arrest was maintained with hypoxanthine or hypoxanthine plus adenosine but reversed the combined inhibitory action of these purines in DOs. Five of six adenosine analogs that bind to purinoceptors demonstrated meiosis-arresting activity but not in relation to their relative affinities for inhibitory or stimulatory adenosine receptors and only at high concentrations. Moreover, in DOs, uridine reversed the inhibitory effect of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, two receptor agonists that are poor substrates for adenosine-metabolizing enzymes. Results of experiments with adenosine kinase inhibitors showed that methylmercaptopurine riboside (MMPR) and tubercidin, but not 5'-amino-5'-deoxyadenosine, reversed meiotic arrest maintained by hypoxanthine +/- adenosine, but this required an additional inhibitory action on de novo purine synthesis. Inhibition of de novo purine synthesis alone was not sufficient because azaserine failed to reverse meiotic arrest. MMPR was a very potent meiosis-inducing agent, completely reversing meiotic arrest in CEOs and DOs in the presence of a variety of meiotic inhibitors. The adenosine deaminase inhibitor deoxycoformycin had opposite effects on oocyte maturation depending on the presence or absence of adenosine: the inhibitory action of hypoxanthine alone was bolstered, but the meiosis-arresting action of adenosine was reversed. These data therefore indicate that at low adenosine concentrations phosphorylation predominates, but at higher adenosine concentrations deaminated products contribute to the meiotic inhibition. This idea was borne out by the ability of inosine to mimic the synergistic interaction of adenosine with hypoxanthine. The action of adenosine is not due to deamination to inosine and conversion to nucleotides through the hypoxanthine salvage pathway because adenosine-mediated inhibition was not compromised in oocytes from mutant mice unable to salvage hypoxanthine.
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PMID:Uptake and metabolism of adenosine mediate a meiosis-arresting action on mouse oocytes. 1033 59

To examine the effect of 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid (TEI-6720), an inhibitor of xanthine oxidase, on purine metabolism in the lung cancer cell line A549, the activities of adenosine deaminase, purine nucleoside phosphorylase, adenine phosphoribosyltransferase, hypoxanthine guanine phosphoribosyltransferase, xanthine oxidase, and guanase together with pyrimidine nucleoside phosphorylase were measured with or without the addition of TEI-6720, and the extracellular concentrations of hypoxanthine, xanthine, inosine, uracil, and uridine were measured after the addition of inosine or uridine to the incubation medium with or without TEI-6720. Moreover, the Na-independent nucleoside transport was determined in A549 cells with or without TEI-6720. TEI-6720 inhibited the activity of xanthine oxidase in A549 cells, but did not affect other enzymes. During incubation, TEI-6720 not only prevented a decrease in the inosine concentration in inosine-containing medium, but also a decrease in the uridine concentration in uridine-containing medium. Furthermore, the Na-independent transport of uridine was inhibited by TEI-6720 with a K(i) value of 4.1 micromol/l. These results indicate that TEI-6720 is an inhibitor of the Na-independent nucleoside transport of uridine and inosine, as well as xanthine oxidase.
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PMID:Effect of TEI-6720, a xanthine oxidase inhibitor, on the nucleoside transport in the lung cancer cell line A549. 1062 41

The induction of apoptosis by adenosine was studied in the mouse neuroblastoma cell line N1E-115. Apoptosis was characterized by fluorescence and electron microscopy, fluorescence-activated cell sorter (FACS) analysis, and caspase activity assays. A sixteen-hour exposure to 100 microM of adenosine led to chromatin condensation and caspase activation. However, selective agonists for all four adenosine receptors were ineffective. Caspase activation could be blocked partially by an inhibitor of the nucleoside transporter, dipyridamole, and completely by uridine, a competing substrate for adenosine transport. 2'-Deoxycoformycin, an inhibitor of adenosine deaminase, enhanced caspase activation by adenosine but had no effect by itself. Caspase activation could be blocked by 5'-amino-5'-deoxyadenosine, which inhibits the phosphorylation of adenosine by adenosine kinase. These results indicate that adenosine receptors are not involved in adenosine-induced apoptosis in N1E-115 cells, but that uptake of adenosine and its subsequent phosphorylation is required.
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PMID:Extracellular adenosine-induced apoptosis in mouse neuroblastoma cells: studies on involvement of adenosine receptors and adenosine uptake. 1122 75


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