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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)

Evaluation of enzyme activities involved in nucleotide metabolism and adenosine production within different cell types can provide important information on their contribution to the overall metabolism of the heart. The following enzyme activities were determined: adenosine kinase (AK), adenosine deaminase (ADA), S-adenosylhomocysteine hydrolase (SAHH), purine nucleoside phosphorylase (PNP), AMP deaminase (AMPD), membrane 5'nucleotidase (M5'N), AMP specific (AC5'N) and IMP specific (IC5'N) cytosolic 5'nucleotidases in (1) rat heart (n = 5), (2) rat cardiomyocytes obtained by collagenase digestion (n = 5), (3) human heart (n = 6) obtained from explants or papillary muscles collected during heart transplantation or mitral valve replacement, and (4) human umbilical cord endothelial cells in primary culture (n = 4). In the human heart, activities (mumol/min/g wet weight) were as follows: AK (0.14 +/- 0.01), ADA (0.46 +/- 0.03), SAHH (0.001 +/- 0.0003), PNP (0.43 +/- 0.08), AMPD (0.41 +/- 0.05), M5'N (1.75 +/- 0.12), IC5'N (0.21 +/- 0.03) and AC5'N (0.11 +/- 0.02). These enzyme activities were lower than those determined in the rat heart with the exception of AC5'N and IC5'N which were equal. The most prominent difference observed was for AMPD and M5'N which were nine and five-fold more active in the rat heart. Rat cardiomyocyte enzyme activities were comparable to those measured in whole rat heart with the exception of ADA (six-fold lower) and PNP (16-fold lower). Endothelial cell activities were notably different from those in the human heart particularly in the case of SAHH (nine-fold higher) and PNP (16-fold higher).(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Cell Cardiol 1994 Nov
PMID:Nucleotide and adenosine metabolism in different cell types of human and rat heart. 789 72

The anti-human immunodeficiency virus agents 2',3'-dideoxyadenosine (ddAdo) and 2'-beta-fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo) are rapidly converted, both in vitro and in vivo, to the corresponding inosine analogs by the widely distributed enzyme adenosine deaminase (EC 3.5.4.4). We have determined the effects of the potent adenosine deaminase inhibitor 2'-deoxycoformycin (2'-dCF) on ddAdo and 2'-beta-F-ddAdo metabolism in MOLT-4 cells and on ddAdo antiviral activity in the ATH8 test system. At levels as low as 5 nM in the incubation medium, 2'-dCF effectively blocks the extracellular deamination of both agents, thus permitting their rapid cellular uptake as the unchanged parent compounds, rather than as the less lipid-soluble 2',3'-dideoxyinosine or 2'-beta-fluoro-2',3'-dideoxyinosine. The result is a significant increase in intracellular levels of the pharmacologically active forms 2',3'-dideoxyadenosine-5'-triphosphate and 2'-beta-fluoro-2',3'-dideoxyadenosine-5'-triphosphate. The effect becomes maximal over the range of 50-250 nM 2'-dCF and declines to control levels when extracellular 2'-dCF levels exceed 1 microM. This decrease in ddAdo and 2'-beta-F-ddAdo phosphorylation with higher levels of the inhibitor appears to result from intracellular penetration of 2'-dCF and consequent inhibition of intracellular deamination, a critical step in the activation of both agents through the 5'-nucleotidase pathway. In anti-human immunodeficiency virus assays, a 2.2-fold increase in ddAdo antiviral potency was seen at 2'-dCF levels of 20 and 50 nM.
Mol Pharmacol 1994 Nov
PMID:Enhancement by 2'-deoxycoformycin of the 5'-phosphorylation and anti-human immunodeficiency virus activity of 2',3'-dideoxyadenosine and 2'-beta-fluoro-2',3'-dideoxyadenosine. 796 62

Here we describe studies of double-stranded RNA (dsRNA) adenosine deaminase in Xenopus laevis, in particular during meiotic maturation, the period during which a stage VI oocyte matures to an egg. We show that dsRNA adenosine deaminase is in the nuclei of stage VI oocytes. Most importantly, we demonstrate that the cytoplasm of stage VI oocytes contains a factor that protects microinjected dsRNA from deamination when dsRNA adenosine deaminase is released from the nucleus during meiotic maturation. Our data suggest that the protection factor is a cytoplasmic dsRNA-binding protein or proteins that bind to dsRNA in a sequence-independent manner to occlude dsRNA from binding to dsRNA adenosine deaminase. The cytoplasmic double-stranded RNA-binding protein(s) does not bind to other nucleic acids and can be titrated at high concentrations of dsRNA. These studies raise the question of whether all dsRNA-binding proteins share endogenous substrates and also suggest potential means of regulating dsRNA adenosine deaminase in vivo.
Mol Cell Biol 1994 Aug
PMID:The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification. 803 19

Transcription arrest plays a key role in the regulation of the murine adenosine deaminase (ADA) gene, as well as a number of other cellular and viral genes. We have previously characterized the ADA intron 1 arrest site, located 145 nucleotides downstream of the transcription start site, with respect to sequence and elongation factor requirements. Here, we show that the optimal conditions for both intron 1 arrest and overall ADA transcription involve the addition of high concentrations of KCl soon after initiation. As we have further delineated the sequence requirements for intron 1 arrest, we have found that sequences downstream of the arrest site are unnecessary for arrest. Also, a 24-bp fragment containing sequences upstream of the arrest site is sufficient to generate arrest downstream of the adenovirus major late promoter only in the native orientation. Surprisingly, we found that deletion of sequences encompassing the ADA transcription start site substantially reduced intron 1 arrest, with no effect on overall levels of transcription. At the same time, deletion of sequences upstream of the TATA box had no significant effect on either process. We believe the start site mutations have disrupted either the assembly or the composition of the transcription complex such that intron 1 site read-through is now favored. This finding, coupled with the increase in overall transcription after high-concentration KCl treatment, allows us to further refine our model of ADA gene regulation.
Mol Cell Biol 1994 Sep
PMID:Control of transcription arrest in intron 1 of the murine adenosine deaminase gene. 806 52

The effects of theophylline upon human alveolar macrophage function were assessed and compared with its action upon macrophage cyclic nucleotide phosphodiesterase (PDE) activity and cyclic adenosine monophosphate (cAMP) levels. In the concentration range of 10 mumol/liter to 1 mmol/liter, theophylline caused a concentration-dependent inhibition of opsonized zymosan-stimulated hydrogen peroxide (H2O2) generation and PDE-catalyzed cAMP hydrolysis and increased the cellular cAMP content. Macrophage H2O2 generation was also inhibited by forskolin, an activator of adenylyl cyclase, but whereas theophylline (1 mmol/liter) and forskolin (1 mumol/liter) exhibited a synergic elevation of macrophage cAMP, there was no synergy between the two agents in the inhibition of respiratory burst. The inhibition of H2O2 generation by theophylline was reversed by the competitive inhibitor of cAMP-dependent protein kinase, (Rp)8-bromoadenosine cyclic 3':5'-monophosphorothioate (Rp-8-Br-cAMPS; 100 mumol/liter), indicating that the functional effect of theophylline was mediated through the elevation of cAMP. The inhibition of H2O2 generation by theophylline was not affected by adenosine deaminase (0.1 U/ml), indicating that the inhibition did not involve adenosine antagonism. It is concluded that theophylline exerts a direct inhibitory action upon human alveolar macrophage function through the elevation of cAMP levels as a result of PDE inhibition, and that this effect is observed at concentrations of theophylline that may be achieved in serum during therapy.
Am J Respir Cell Mol Biol 1994 May
PMID:Theophylline suppresses human alveolar macrophage respiratory burst through phosphodiesterase inhibition. 817 21

Pluripotency of isolated rabbit inner cell masses (ICMs) and cultured (3 days) inner cell mass (ICM) cells was tested by injecting these donor cells into day 3.5 blastocysts (experiment 1) or day 3 morulae (experiment 2) to produce chimeric embryos. Injected (n = 107) and noninjected (n = 103) embryos were transferred to the opposite uterine horns of the same recipient females. Chimerism was determined by adenosine deaminase (ADA) isozyme analysis on fetal tissue and by eye pigmentation at midgestation. In experiment 1, 53% and 64%, respectively, of blastocysts injected with ICMs or cultured ICM cells developed to midgestation, compared with 52% and 48% for controls. Of these fetuses, four (31%) and one (6%), respectively, had ADA chimerism. In experiment 2, 38% and 62%, respectively, of the morulae injected with ICMs or cultured ICM cells developed to midgestation, compared with 46% and 56% for control morulae. Six (43%) chimeric fetuses from morulae injected with ICMs were detected by ADA analysis, but 12 (86%) chimeric fetuses were detected by eye pigmentation, indicating that eye pigmentation was a more sensitive marker for chimerism than our ADA assay. None of the 14 fetuses recovered after injecting morulae with cultured ICM cells were chimeric with either marker. No chimeras developed from control embryos. These studies demonstrate 1) that pregnancy rates are not compromised by injection of blastocysts or morulae with ICMs or cultured ICM cells, 2) that chimeric rabbit fetuses can be produced by injecting ICMs into either blastocysts or morulae, and 3) that cultured ICM cells can contribute to embryonic development when injected into blastocysts.
Mol Reprod Dev 1993 Oct
PMID:Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae. 825 63

Tissue injury associated with myocardial ischemia is assumed to largely result from the toxic effects of active oxygen species generated by accumulated polymorphonuclear leukocytes (PMNs). Recent reports have indicated that adenosine can interfere with the PMN function in vitro. The potential of adenosine to influence PMN-mediated myocardial tissue injury was assessed using a model of ischemia-reperfusion injury developed in the isolated working guinea-pig heart perfused with homologous PMNs. After an initial work phase, hearts were subjected to 30 min low-flow ischemia (1 ml/min) in the absence and presence of PMNs. Work was resumed after 15 min reperfusion in a non-working mode (Langendorff). Adenosine in the coronary effluent reached a maximum of 0.2 microM during low-flow ischemia. Recoveries of external heart work and cardiac output were reduced from about 80% to about 40% by PMNs. Infusion of adenosine deaminase (ADA, 5 U/ml), theophylline (50 microM) or the selective A1-antagonist dipropyl-8-cyclopentylxanthine (0.1 microM) prevented this effect. Furthermore, application of adenosine (0.1 microM) in combination with PMNs also resulted in a loss of pump function, even in the absence of a direct ischemic stimulus. The data indicate that adenosine contributes to post-ischemic, PMN-mediated damage in the isolated working guinea-pig heart model by a receptor-mediated action.
J Mol Cell Cardiol 1993 Aug
PMID:Adenosine contributes to neutrophil-mediated loss of myocardial function in post-ischemic guinea-pig hearts. 826 62

We have solved the structure of Escherichia coli cytidine deaminase (CDA) complexed to the transition state analog, 5-fluoroprimidin-2-one riboside. The monomer of the alpha 2 CDA dimer is composed of a small N-terminal alpha-helical domain with no obvious connection to the active sites, and two, larger, core domains. The two core domains have nearly identical tertiary structures and are related by approximate 2-fold symmetry, but lack internal amino acid sequence homology. Comparison of the core domain structure with known structures by sequence homology and structural compatibility searches suggests that the CDA tertiary structure cannot be superimposed on any known protein structure. The two active sites per dimer are formed across the subunit interface. The N-terminal core domain provides a pyrimidine nucleoside and zinc-binding pocket and the structurally homologous C-terminal core domain in the other monomer covers this active-site cleft, completely sequestering the ligand from solvent. The deeply buried zinc-binding site is formed by a novel "topological switch point" at the amino termini of two alpha-helices in consecutive alpha-beta-alpha-beta segments. The transition state analog is bound as a covalent hydrate at C4. The inhibitor hydroxyl oxygen atom interacts both with the zinc atom and the Glu104 carboxylate group, affording high differential affinity for the hydroxyl group relative to a hydrogen atom, in a manner reminiscent of that observed in adenosine deaminase (ADA). Unlike the latter enzyme, the zinc atom is coordinated in a tetrahedral ligand field to two cysteine and one histidine ligands, plus the hydroxyl group. Moreover, the inhibitor stereochemistry is of the opposite hand from that of the corresponding ADA inhibitor at C4(R), but is the same at the hydroxyl group O4(S). A consequence of these stereochemical differences is that in CDA a single conserved carboxylate side-chain, Glu104, can provide all of the necessary proton transfer functions involved in generating the zinc hydroxide nucleophile, and protonating the pyrimidine ring nitrogen atom and leaving amino group. The differences in zinc ligands, ligand-binding stereochemistry, and tertiary structures of CDA and ADA strongly suggest that the common features of transition state stabilization arose by convergent evolution.
J Mol Biol 1994 Jan 14
PMID:Cytidine deaminase. The 2.3 A crystal structure of an enzyme: transition-state analog complex. 828 86

Recent work has suggested that chronic ethanol treatment induces heterologous desensitization of adenylate cyclase in a number of cell lines maintained in culture and that this phenomenon is mediated by adenosine. It has been proposed that ethanol induces the accumulation of extracellular adenosine, which then down-regulates the Gs alpha protein and leads to heterologous desensitization. Here we investigated the effects of chronic ethanol treatment on the expression of Gs alpha, Gi alpha, and Go alpha, as well as cAMP signal transduction, in NG108-15 cells and further examined the role of adenosine in mediating these effects. Pretreatment of NG108-15 cells with 200 mM ethanol for 2 days reduced membrane levels of Gs alpha and Gi alpha and increased those of Go alpha. However, ethanol did not reduce the levels of Gs alpha and Gi alpha 2 mRNA in these cells. The ability of ethanol to alter alpha subunit expression was not reversed by removal of extracellular adenosine and could not be mimicked by an adenosine agonist. Chronic ethanol treatment increased both basal and agonist-stimulated cAMP accumulation in NG108-15 cells. Whereas the increase in basal cAMP was abolished by acute addition of adenosine deaminase, the increase in agonist-stimulated cAMP accumulation was not. Morphological examination of the cells indicated that ethanol inhibited cell division and promoted the apparent differentiation of the cells. These results indicate that ethanol induces complex alterations in guanine nucleotide-binding protein alpha subunit expression and cAMP signal transduction in NG108-15 cells and that it is unlikely that these effects are mediated simply by adenosine.
Mol Pharmacol 1993 Feb
PMID:Ethanol differentially regulates guanine nucleotide-binding protein alpha subunit expression in NG108-15 cells independently of extracellular adenosine. 838 6

A 4-kb HindIII fragment that supported the efficient autonomous replication of plasmid vector pDY-, a replication-defective construct based on Epstein-Barr virus sequences, in human K562 cells was rescued from amplified double-minute chromosomes containing the murine adenosine deaminase locus. Polymerase chain reaction assays of size-fractionated nascent strands demonstrated that replication initiation occurred within the same 1- to 2-kb region of this fragment in autonomously replicating plasmids containing the sequence in either orientation, in double-minute chromosomes, and in the single-copy locus at its normal chromosomal location. The complete sequence of this fragment was determined; it contains a 248-bp polypurine tract and consensus binding site sequences for several putative transcription and replication factors.
Mol Cell Biol 1993 Oct
PMID:Analysis of a replication initiation sequence from the adenosine deaminase region of the mouse genome. 841 98


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