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)

Previous work has shown that platelet-derived adenine nucleotides modulate neutrophil superoxide anion (O2-) generation. Additional studies were undertaken to characterize the effects of authentic adenosine (ADO) and its nucleotide derivatives on the inflammatory functions of human neutrophils. Stimulus-specific inhibition of neutrophil O2- generation by ADO in response to FMLP was verified. In addition, the ability of ATP, ADP, and AMP to limit neutrophil O2- generation induced by FMLP (0.2 to 0.5 microM) was demonstrated. The concentration producing 50% inhibition for nucleotide inhibition of neutrophil O2- generation was in the rank order of ADO (0.1 microM) less than AMP (0.5 microM) less than ADP less than or equal to ATP (5 microM). Guanine and inosine nucleotides (0.01 to 100 microM) did not inhibit FMLP-stimulated neutrophil O2- generation. Neutrophil degranulation in response to FMLP was only modestly inhibited by adenine nucleotides and ADO. Adenosine and ADP failed to affect chemotaxis of neutrophils stimulated with FMLP. The inability of non-metabolizable analogs to mimic the inhibitory effects of authentic ATP or ADP on the neutrophil O2- response suggested that metabolism of added nucleotides is necessary for their effectiveness. Both TLC and HPLC confirmed that ATP and ADP were converted to AMP and ADO after their incubation with unstimulated or FMLP-activated neutrophils. The addition of adenosine deaminase to neutrophil reaction mixtures in which conversion of added nucleotides was apparent removed detectable ADO but failed to completely abrogate the inhibition of neutrophil O2- generation by accumulated AMP. The kinetics of inhibition of FMLP-induced neutrophil O2- generation by ATP and ADP also indicated that conversion of these nucleotides to ADO and/or AMP may be essential for their ability to reduce neutrophil responses.
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PMID:Regulation of human neutrophil functions by adenine nucleotides. 253 67

The effect of adenosine on the metabolism of prelabelled adenine nucleotides was investigated in concanavalin-A-stimulated rat lymphocytes. Adenosine in the presence of the adenosine deaminase inhibitor, deoxycoformycin, caused a 2-fold increase in the ATP concentration. This effect was, in part, countereacted by an increased rate of adenine nucleotide catabolism, which could be explained by a stimulation of AMP deaminase (EC 3.5.4.6). At the same time a continuous rate of labelled adenosine production was found, which was not affected by the increased ATP concentration and which could only be detected by the trapping effect of a high concentration of added unlabelled adenosine. It is concluded that the rate of the substrate cycle between AMP and adenosine is low (1.9 +/- 0.2 nmol/h per 10(7) cells) in comparison to the rate of AMP deamination.
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PMID:The rate of the AMP/adenosine substrate cycle in concanavalin-A-stimulated rat lymphocytes. 255 90

AMP-sepharose 4B has been widely used as a general ligand affinity chromatography for purification of AMP deaminase, 5'-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AMP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a single run. Using 0.5 M NaCl, 10 mM ATP and 5 mM adenosine as eluting agents, it was possible to separate on AMP-sepharose column AMP deaminase "high Km" and "low Km" 5'-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, "high Km" and "low Km" 5'-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and "high Km" 5'-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 mumols/min/mg protein of AMP deaminase, "high Km" 5'-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.
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PMID:The application of affinity chromatography for the separation of "high Km" and "low Km" 5'-nucleotidase and other AMP metabolizing enzymes. 255 31

We compared the response of rat PC12 cells and a derivative PC18 cell line to the effects of adenosine receptor agonists, antagonists, and adenine nucleotide metabolizing enzymes. We found that theophylline (an adenosine receptor antagonist), adenosine deaminase, and AMP deaminase all decreased basal cyclic AMP content and tyrosine hydroxylase activity in the PC12 cells, but not in PC18 cells. Both cell lines responded to the addition of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, adenosine receptor agonists, by exhibiting an increase in tyrosine hydroxylase activity and cyclic AMP content. The latter finding indicates that both cell lines contained an adenosine receptor linked to adenylate cyclase. We found that the addition of dipyridamole, an inhibitor of adenosine uptake, produced an elevation of cyclic AMP and tyrosine hydroxylase activity in both cell lines. Deoxycoformycin, an inhibitor of adenosine deaminase, failed to alter the levels of cyclic AMP or tyrosine hydroxylase activity. This suggests that uptake was the primary inactivating mechanism of adenosine action in these cells. We conclude that both cell types generated adenine nucleotides which activate the adenosine receptor in an autocrine or paracrine fashion. We found that PC12 cells released ATP in a calcium-dependent process in response to activation of the nicotinic receptor. We also measured the rates of degradation of exogenous ATP, ADP, and AMP by PC12 cells. We found that the rates of metabolism of the former two were at least an order of magnitude greater than that of AMP. Any released ATP would be rapidly metabolized to AMP and then more slowly degraded to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Adenosine receptor activation and the regulation of tyrosine hydroxylase activity in PC12 and PC18 cells. 257 81

Deoxyadenosine is known to be toxic to both proliferating and resting lymphocytes that lack adenosine deaminase (ADA) activity. We now show that human monocytes are also highly sensitive in vitro to nanomolar concentrations of deoxyadenosine plus the ADA inhibitor deoxycoformycin, and to the ADA-resistant analogue 2-chlorodeoxyadenosine (CdA). Monocytes exposed to deoxyadenosine or to CdA in vitro accumulate massive DNA damage detectable within 1 hour. The DNA damage in monocytes exposed to CdA is associated with a decrease in protein synthesis and with inhibitions of phagocytosis and IL-6 secretion. However, unlike lymphocytes with similar DNA damage, the monocytes show no significant NAD or ATP depletion until cell viability declines. The selective toxicity of CdA to monocytes was confirmed by in vivo studies. In almost all patients receiving CdA infusion chemotherapy for cutaneous lymphoma, the blood monocytes counts fell to near 0 during one week of therapy. Our results suggest that CdA and related compounds may have potential clinical use in the therapy of immune disorders associated with monocyte/macrophage activation.
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PMID:Profound toxicity of deoxyadenosine and 2-chlorodeoxyadenosine toward human monocytes in vitro and in vivo. 257 48

Deoxyadenosine has been implicated as the toxic metabolite causing profound lymphopenia in immunodeficient children with a genetic deficiency of adenosine deaminase (ADA), and in adults treated with the potent ADA inhibitor deoxycoformycin. However, the biochemical basis for deoxyadenosine toxicity toward lymphocytes remains controversial. The present experiments have examined in detail the sequential metabolic changes induced in nondividing human peripheral blood lymphocytes by incubation with deoxyadenosine plus deoxycoformycin, or with 2-chlorodeoxyadenosine (CdA), an ADA resistant deoxyadenosine congener with anti-leukemic and immunosuppressive properties. The lymphotoxic effect of deoxyadenosine and CdA required their phosphorylation, and was inhibited by deoxycytidine. As early as 4 h after exposure to the deoxynucleosides, strand breaks in lymphocyte DNA began to accumulate, and RNA synthesis decreased. These changes were followed by a significant fall in intracellular NAD levels at 8 h, a drop in ATP pools at 24 h, and cell death by 48 h. Incubation of the lymphocytes with 5 mM nicotinamide, a NAD precursor and an inhibitor of poly(ADP-ribose) synthetase, prevented NAD depletion. The nicotinamide treatment also rendered the lymphocytes highly resistant to deoxyadenosine and CdA toxicity, without altering dATP formation or the accumulation of DNA strand breaks. The poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide exerted a similar although less potent effect. These results suggest that NAD depletion, probably triggered by poly(ADP-ribose) formation, is the principle cause of death in normal resting human lymphocytes exposed to deoxyadenosine plus deoxycoformycin, or to CdA.
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PMID:Mechanism of deoxyadenosine and 2-chlorodeoxyadenosine toxicity to nondividing human lymphocytes. 257 98

Dopamine and 2-chloroadenosine independently promoted the accumulation of cyclic AMP in retinas from 16-day-old chick embryos. The two compounds added together either in saturating or subsaturating concentrations were not additive for the accumulation of the cyclic nucleotide in the tissue. This fact was shown to be due to the existence of an adenosine receptor that mediates the inhibition of the dopamine-dependent cyclic AMP accumulation in the retina. Adenosine inhibited, in a dose-dependent fashion, the accumulation of cyclic AMP induced by dopamine in 12-day-old chick embryo retinas, with an IC50 of approximately 1 microM. This effect was not blocked by dipyridamole. N6-(l-Phenylisopropyl)adenosine, (l-PIA) was the most potent adenosine analog tested, showing an IC50 of 0.1 microM which was two orders of magnitude lower than its stereoisomer d-PIA (10 microM). The maximal inhibition of the dopamine-elicited cyclic AMP accumulation by adenosine and related analogs was 70%. The inhibitory effect promoted by adenosine was blocked by 3-isobutyl-1-methylxanthine (IBMX) or by adenosine deaminase. Adenine was not effective; whereas ATP and AMP promoted the inhibition of the dopamine effect only at very high concentrations. Apomorphine was only 30% as effective as dopamine in promoting the cyclic AMP accumulation in retinas from 11- to 12-day-old embryos and 2-chloroadenosine did not interfere with the apomorphine-mediated shift in cyclic AMP levels. In the retinas from 5-day-old posthatched chickens dopamine and apomorphine were equally effective in eliciting the accumulation of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of A1 adenosine receptors modulating dopamine-dependent cyclic AMP accumulation in the chick embryo retina. 257 99

Adenosine inhibition of hormone-sensitive adenylate cyclase activity was investigated using isolated myocardial membranes prepared from rat hearts. When cyclase activity was determined in membranes, using [alpha-32P]ATP as substrate, 10(-5) M adenosine inhibited isoproterenol-stimulated adenylate cyclase activity by 25% but did not inhibit basal activity or fluoride (5 mM) activation of the enzyme. The adenosine reduction of isoproterenol-sensitive cyclase activity was dependent on GTP but was not prevented by 10(-3) M theophylline. Adenosine neither appeared to compete with ATP for the substrate converting site of the enzyme nor reduced 5'-guanylyl imidodiphosphate activation of the enzyme. Inasmuch as lower concentrations of adenosine had no influence on enzyme activity, endogenous adenosine may be present in the adenylate cyclase assay. To obviate the effects of endogenous adenosine, the adenylate cyclase assay was then modified to a 2'-deoxy system with [alpha-32P]dATP used as the substrate in the presence of adenosine deaminase. With this assay system, the 15% inhibition of isoproterenol-stimulated adenylate cyclase activity produced by the adenosine receptor agonists, 10(-8) M 2-chloroadenosine or phenylisopropyladenosine, was prevented by 10(-4) M 8-phenyltheophylline or isobutylmethylxanthine (IBMX), respectively. While under these assay conditions, 10(-7) M 2',5'-dideoxyadenosine, a P-site analogue, did not influence the hormone-sensitive cyclase activity. The 35% reduction of the hormone-sensitive enzyme produced by this analogue at 10(-5) M was not prevented by IBMX. These results suggest that nanomolar concentrations of adenosine analogues interact with a methylxanthine-sensitive adenosine receptor that mediates the attention of membrane hormone-sensitive adenylate cyclase activity.
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PMID:Adenosine inhibition of catecholamine-stimulated cardiac membrane adenylate cyclase. 258 60

A kindred with an autosomal dominant form of chronic hemolytic anemia has been found to have a 40- to 70-fold elevation in erythrocyte adenosine deaminase (ADA) activity in association with depletion of red blood cell (RBC) ATP pools. ADA activities in B lymphoblasts, skin fibroblasts, and granulocytes were normal. There were no alterations in the kinetic properties of partially purified proband ADA. We have shown by Western blot analysis that the elevation in ADA activity is accompanied by a corresponding increase in the amount of immunoreactive ADA protein. Southern blot analysis of proband DNA ruled out gene amplification and revealed no gross insertions, deletions, or rearrangements in the ADA gene. Northern blot analysis demonstrated a marked increase in the amount of ADA mRNA in proband and sibling reticulocytes compared to high reticulocyte controls. ADA mRNA levels in B lymphoblasts from the proband, sibling, and GM558 cell line were normal. Cloning and sequencing of proband reticulocyte cDNA revealed normal ADA mRNA sequence. No polymorphisms were detected among the seven clones studied. RNase mapping of the 5'- and 3'-non-coding sequences confirmed the quantitative increase in reticulocyte ADA mRNA and verified that these regions were normal in length and sequence. Southern blot analysis of DNA from four affected and three unaffected family members revealed two restriction fragment length polymorphisms (RFLPs) which segregate with the ADA allele from the unaffected grandfather. Both RFLPs are present in the unaffected grandchild and absent in the affected grandchild. These findings are consistent with a cis- mutation within the ADA gene, but they do not rule out a trans- mutation affecting some non-ADA regulatory factor. We conclude that erythrocyte-specific ADA overproduction is associated with increased amounts of structurally normal ADA mRNA. This increase may result from either increased transcription of the ADA gene or altered post-transcriptional processing resulting in increased stability of the RNA transcript. Further elucidation of the defect should provide valuable insights into the normal tissue-specific regulation of the ADA gene and the mechanisms by which erythroid cells regulate gene expression during differentiation.
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PMID:Erythrocyte-specific overproduction of adenosine deaminase: molecular genetic studies. 262 27

2,6-Diaminopurine (DAP) and 2,6-diaminopurine 2'-deoxyriboside (DAPdR) are analogs of adenine and deoxyadenosine, respectively. It was the purpose of this study to compare these analogs under identical conditions in order to define their inhibitory properties and the underlying mechanism in L1210 mouse leukemia cells. In a 5-day cell growth experiment, DAP exerted a significantly stronger antiproliferative effect than DAPdR. Correspondingly, colony formation of L1210 cells in soft agarose was inhibited by DAP to a greater extent than by DAPdR. A differential distribution of L1210 cells in the cell cycle resulted from an exposure to DAP and DAPdR. While DAPdR arrested cells in the G1/G0 phase of the cell cycle, DAP appeared to lead to an accumulation of G2/M cells. The diaminopurines were combined with modulatory agents to test the antiproliferative action of the combinations. Deoxycytidine partially rescued the cells from the growth inhibitory action of DAPdR without affecting the growth of DAP-treated cells. When adenine was used, the antiproliferative effect of DAPdR was slightly enhanced while the effect of DAP was completely abolished. 8-Aminoguanosine, a specific inhibitor of purine nucleoside phosphorylase, synergistically potentiated the cytostatic effect of DAPdR. However, this inhibitor did not alter DAP effects. At the biochemical level, the target of DAPdR was ribonucleotide reductase which was in line with a drastic expansion of the dGTP pool in DAPdR-treated cells. In cells exposed to DAP, high levels of DAP riboside triphosphate were measured; concomitantly, the ATP level dropped markedly. Enzymological studies revealed that DAPdR is an excellent substrate of adenosine deaminase giving rise to the formation of deoxyguanosine. DAP was found to be activated in the purine nucleoside phosphorylase reaction and in a phosphoribosyl-pyrophosphate-dependent reaction. The data from this comparative study suggest that DAPdR and DAP possess different toxicity mechanisms. DAPdR and DAP possess different toxicity mechanisms. DAPdR acts as a precursor of deoxyguanosine, and DAP is metabolically activated to DAP-containing ribonucleotide analogs. These different metabolic routes seem to account for the different effects of DAP and DAPdR at the cellular level.
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PMID:Metabolic activation of 2,6-diaminopurine and 2,6-diaminopurine-2'-deoxyriboside to antitumor agents. 262 71


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