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Query: UNIPROT:P06889 (Mol)
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A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplified copies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clones encompassing the entire ADA structural gene were isolated from this genomic library and used for subsequent structural and functional analyses. Nuclease protection and primer extension analyses served to identify the location of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity was found by functional analyses to reside within a 240-base-pair fragment which contains the transcription initiation sites. Sequences upstream of the transcription initiation sites are very G + C rich (77%) and include a 22 nucleotide stretch of deoxyguanylate residues and two potential Sp1 transcription factor-binding sites. Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that are highly conserved with regard to both sequence content and location and may represent genetic elements which are involved in ADA gene expression.
Mol Cell Biol 1986 Dec
PMID:Molecular cloning of the murine adenosine deaminase gene from a genetically enriched source: identification and characterization of the promoter region. 243 2

The isolated perfused rat heart was used to study the influence of adenine nucleotides and their metabolites on vulnerability to ventricular fibrillation. In this model the incidence of ventricular arrhythmias after coronary artery ligation is determined by the extracellular K+ concentration; with perfusate K+ of 2.0 and 3.0 mmol/l hearts develop a high incidence of ventricular arrhythmias and fibrillation while arrhythmias are not encountered with perfusate K+ of 9.0 mmol/l. Assay of adenine nucleotides in uninvolved and ischaemic myocardium of these hearts showed a direct relationship between incidence of ventricular fibrillation and tissue levels of cyclic AMP but not tissue levels of lactate, high energy phosphates, adenosine, inosine and hypoxanthine/xanthine. Administration of dibutyryl cyclic AMP to isolated rat hearts reduced the ventricular fibrillation threshold; this action of cyclic AMP was effectively antagonized by adenosine and its N-ethylcarboxamido analogue but not by 2-chloroadenosine, phenylisopropyladenosine, cyclohexyladenosine and the adenosine deaminase inhibitor, EHNA. 2-Chloroadenosine, like adenosine, inhibited the increase in heart rate caused by DBcAMP. All the adenosine analogues had antiarrhythmic activity against spontaneously occurring ventricular arrhythmias during coronary artery occlusion. Adenosine analogues also antagonized the effect of dibutyryl cyclic AMP whereby it prolongs the QT interval. Adenosine, by as yet incompletely defined mechanisms, may act as an antagonist to the cyclic AMP mediated increase in vulnerability which contributes to the genesis of ventricular fibrillation in the early phase of myocardial ischaemia.
J Mol Cell Cardiol 1987 Oct
PMID:Adenine nucleotides and ventricular fibrillation. 244 89

Neplanocin A [(-)-9-[trans-2',trans-3'-dihydroxy-4'-(hydroxymethyl)-cyclopent-4 '- enyl]-adenine] and 9-[trans-2',trans-3'-dihydroxycyclopent-4'-enyl]-adenine (1) and -3-deazaadenine (2) are potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) in mouse L929 cells. When cells were treated for 15 min with varying concentrations of the drugs, the IC95 values (concentration needed to produce 95% inhibition of AdoHcy hydrolase) for neplanocin A, 1, and 2 were determined to be 0.2 microM, 0.5 microM, and 0.5 microM, respectively. Incubation of L929 cells with 1.0 microM concentrations of neplanocin A, 1, or 2 produced rapid inactivation of AdoHcy hydrolase (within 30 min the enzyme was 95% inhibited), which persisted for at least 72 hr. At lower concentrations (0.032 microM), substantial recovery of AdoHcy hydrolase activity was noted after 48 and 72 hr in cultures treated with neplanocin A but not in cultures treated with 1 or 2. L929 cells treated with neplanocin A, 1 or 2 showed a rapid increase in intracellular levels of AdoHcy (as well as the ratio of AdoHcy/S-adenosylmethionine). Cells treated with neplanocin A also contained significant amounts of S-neplanocylmethionine, whereas cells treated with 1 or 2 showed no evidence of the formation of a similar metabolite. When neplanocin A and adenosine were incubated in cell lysates, rapid conversion to neplanocin D and inosine, respectively, were observed, illustrating the affinity of these nucleosides for cellular adenosine deaminase. In contrast, when 1 and 2 were incubated in cell lysates, no evidence for deamination was observed. These data illustrate that compounds 1 and 2 retain the inhibitory activity of neplanocin A toward cellular AdoHcy hydrolase, producing elevated cellular levels of AdoHcy. However, by removing the 4'-hydroxymethyl group from neplanocin A, analogs 1 and 2 are no longer substrates for adenosine deaminase and adenosine kinase.
Mol Pharmacol 1988 Jun
PMID:Effects of 9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-adenine and -3-deazaadenine on the metabolism of S-adenosylhomocysteine in mouse L929 cells. 245 88

The adenosine deaminase (ADA) locus appears to be under complex transcriptional control since levels of ADA enzyme activity vary greatly between different tissues and stages of development. Evidence that a trans-acting factor may be involved with the regulation of this locus came from previous experiments where fusion of ADA-negative human JEG cells and mouse ADA-positive cells led to the trans-activation of human ADA in a hybrid nucleus. Here, we demonstrate that the near euploid mouse embryo fibroblast cell line, CAK, also lacks detectable ADA enzyme activity due to altered gene regulation. We further demonstrate that ADA in CAK cells is not amenable to activation by somatic cell fusion. Following treatment with 5-azacytidine and Xyl-A selection (for ADA), however, CAK clones were obtained that stably express the ADA gene. Molecular analysis of the parental CAK cells and the ADA-positive derivative clones demonstrated that both 5' and 3' regions of the ADA gene had become hypomethylated in the ADA+ clones. We conclude that methylation is another element involved with the transcriptional control of the ADA gene and that ADA might serve as a useful model for studying the interaction of cis- and trans-acting regulational elements.
Somat Cell Mol Genet 1989 Jan
PMID:Hypomethylation and ADA gene expression in mouse CAK cells. 246 55

A quantum chemical study of adenosine, formycin, and their 2-NH2 and 2-F derivatives is performed. The tautomerism of neutral and protonated species as well as the protonation of adenosine, formycin, and their derivatives are theoretically studied using semiempirical MNDO and AM1, as well as ab initio STO-3G methods. Calculations have been performed on a reduced model, in which the ribose moiety has been substituted by a hydroxy-methyl group. Results indicate that adenosine is mainly protonated at the N1 atom, whereas formycin can be protonated on N1 or N3, depending on the tautomeric form (N8-H or N7-H). The quantum chemical study of the N1-protonated molecules shows that a second protonation of adenosine is mainly on the N3 atom, whereas formycin can be protonated on N8 or N3, depending on the tautomeric form. On the other hand, results indicate that the protonation of formycin and its derivatives at the N1 atom leads to a change in their tautomeric preference from N7-H to N8-H. The importance of both tautomerism and protonation reactions in the mechanism of action of adenosine deaminase is studied by means of a quantitative structure activity relationships strategy. Significant correlations were found between several electronic parameters and the logarithm of the maximum rate of deamination (log Vm) of the studied compounds. For formycin and its derivatives, it was necessary to consider their N8-H tautomeric forms. The electronic parameters giving good correlations were as follows: energy of the minimum of the ab initio molecular electrostatic potential on N1, net charge over purine (pyrazolo-pyrimidine) and pyrimidine rings, and the N1 protonation energy. It must be noted that all these parameters are informative in relation to a proton attack. Adenosine and purine ribosides have been studied largely because of their high biological relevance. They are constituents of nucleic acids, intermediates in secondary metabolism, neuromodulators, and neurohormones. Their analogues have been extensively used because of their wide range of pharmacological effects (1). Formycin A (Fig. 1) is one of the most studied analogues of adenosine. It is a natural product extracted from Nocardia interforma (2) with proven antiviral (3-5), antibiotic (2), immunodepressant (6), antitumor (6), and antimetabolic (5) activities.
Mol Pharmacol 1989 Feb
PMID:Theoretical study of the protonation and tautomerization of adenosine, formycin, and their 2-NH2 and 2-F derivatives: functional implications in the mechanism of reaction of adenosine deaminase. 253 60

We have recently shown the presence of adenosine receptors coupled to adenylate cyclase in anterior pituitary and in the present studies we have investigated the effects of adenosine on ACTH release. The 'R'-site specific analogs of adenosine such as N-Ethylcarboxamide adenosine (NECA), L-N6-phenylisopropyl adenosine (PIA), 2-chloro-adenosine (2-Cl-Ado) all stimulated ACTH release in a dose-dependent manner. NECA was the most potent analog and stimulated ACTH release by about 170% with an apparent Ka of 0.1 microM, whereas PIA and 2-Cl-Ado were less potent and stimulated the release by about 110% and 125% with an apparent Ka of 0.2 and 0.4 microM respectively. The stimulation of ACTH release by NECA was inhibited by 3-isobutyl-1-methylxanthine (IBMX). On the other hand, adenosine deaminase (ADA) treatment of the cells also stimulated ACTH release as well as adenylate cyclase activity by about 2-fold, suggesting that endogenous adenosine plays an inhibitory role in the release of ACTH. Other agents, such as corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and forskolin (FSK) also stimulated ACTH release from these cells. In addition, the stimulation by an optimal concentration of NECA was almost additive with maximal stimulation caused by VIP and FSK. These data suggest that adenosine modulates ACTH release from anterior pituitary through its interaction with adenosine receptors coupled to adenylate cyclase.
Mol Cell Biochem 1989 Aug 15
PMID:Adenosine regulates the release of adrenocorticotropic hormone (ACTH) from cultured anterior pituitary cells. 255 Jul 86

Recent evidence suggests that ethanol initially causes an increase in receptor-dependent cAMP levels, followed by heterologous desensitization of receptors coupled to GS after chronic exposure. Here we investigated the role of adenosine in mediating these responses. We found that ethanol caused accumulation of extracellular adenosine in NG108-15 and S49 lymphoma cells. This adenosine activated adenosine receptors to increase intracellular cAMP levels. The addition of adenosine deaminase, to degrade accumulated extracellular adenosine, or isobutyl-methylxanthine, an adenosine receptor antagonist, completely blocked ethanol-induced increases in cAMP levels in NG108-15 cells. Chronic exposure of NG108-15 and S49 wild type cells to ethanol resulted in heterologous desensitization of adenosine receptor- and prostaglandin E1 receptor-dependent cAMP signal transduction. Coincubation of NG108-15 and S49 wild type cells with adenosine deaminase and ethanol for 48 hr prevented heterologous desensitization. Moreover, mutant S49 cells, which are unable to transport adenosine, did not accumulate extracellular adenosine after incubation with ethanol and did not develop ethanol-induced heterologous desensitization. Our results suggest that adenosine is an important mediator of both the acute and chronic effects of ethanol on cAMP signal transduction.
Mol Pharmacol 1989 Nov
PMID:Adenosine is required for ethanol-induced heterologous desensitization. 255 72

Adenosine deaminase is found primarily in the cytoplasm of many cell types. In the human erythrocyte, about 30 per cent of the total adenosine deaminase activity is membrane associated, and about two-thirds of this is inactivated by treatment of intact erythrocytes with the nonpenetrating reagent diazotized sulfanilic acid, without affecting lactate dehydrogenase, a soluble cytoplasmic enzyme. This indicates that within the cell membranes, the catalytic site of about two-thirds of the adenosine deaminase faces the external medium, i.e., ecto adenosine deaminase. Localization of adenosine deaminase activity at the cell membrane is demonstrated directly by electron microscopy by use of the substrate 6-Chloropurine ribonucleoside, which is dechlorinated by adenosine deaminase to produce Cl-, which is precipitated at its locus of formation by added Ag+, and the precipitated AgCl converted into the electron dense Ag0 upon exposure to light. From the Hydropathic Profile of the amino acid sequence of adenosine deaminase it is evident that there are two hydrophobic domains of sufficient length to span a biological membrane, and it is proposed that these domains could function to anchor the enzyme to the membrane. The importance of adenosine deaminase is indicated by the fatal immuno-deficiency which results from untreated genetic adenosine deaminase deficiency. It may be important to determine whether the amount of ecto adenosine deaminase activity is better suited to assess the clinical status of adenosine deaminase deficient patients that the currently used total cellular enzyme activity.
Mol Cell Biochem 1989 Apr 11
PMID:Ecto-enzyme activity of human erythrocyte adenosine deaminase. 277 Jul 11

The S-adenosylhomocysteine (SAH) hydrolase inhibitor adenosine dialdehyde was used in isolated guinea pig hearts to determine the contribution of the transmethylation pathway to cardiac adenosine formation. This inhibitor did not alter cardiac hemodynamics but effectively inhibited SAH-hydrolase activity under in vitro and in vivo conditions. In normoxic perfused hearts adenosine dialdehyde (10 microM) caused tissue levels of SAH to linearly increase at a rate of 160 pmol/g/min over 60 min. At the same time adenosine dialdehyde decreased release of adenosine into the coronary effluent perfusate by 16 pmol/min (34%). Hypoxic perfusion (30% O2) of guinea-pig hearts increased release of adenosine from 43 to 3700 pmol/min. However, rate of SAH formation in the presence of adenosine dialdehyde was only slightly enhanced from 160 to 200 pmol/g/min and adenosine dialdehyde did not significantly alter the hypoxia induced adenosine release. Since all experiments were performed in the presence of the adenosine deaminase inhibitor EHNA (5 microM) the results demonstrate: (1) the transmethylation pathway of the heart contributes one third to global cardiac adenosine production under normoxic conditions and provides a constant source of adenosine independent of tissue oxygenation. (2) The majority of SAH-derived adenosine is salvaged most likely via adenosine kinase. (3) The hypoxia induced adenosine production is predominantly derived from enhanced 5' AMP hydrolysis.
J Mol Cell Cardiol 1989 Aug
PMID:Contribution of S-adenosylhomocysteine to cardiac adenosine formation. 277 14

Adenosine deaminase-deficient mutants of a mouse lymphoma cell line S49 have been isolated by a two-step selection process. In the first step, we derived mutant lines containing haploid levels of adenosine deaminase activity from wild-type cells. The selective medium contained tritiated deoxyadenosine, deoxycytidine, and deoxycoformycin. Wild-type cells were killed, presumably because of suicidal incorporation of tritiated deoxyadenosine via the adenosine deaminase pathway. The second step was to derive, from the partially deficient mutants, sublines that were virtually lacking adenosine deaminase, using tritiated deoxyadenosine and deoxycytidine. Four mutant clones were found to contain less than 5% of the enzyme activity of wild-type cells and virtually no immunoreactive adenosine deaminase protein. Northern blot analysis showed that the levels of adenosine deaminase mRNA were drastically reduced. Back-selection for adenosine deaminase-positive revertants can be accomplished by using a medium containing deoxyadenosine (as a sole source of purine), aminopterin, and thymidine or, alternatively, by using deoxyadenosine alone in a serum-free medium.
Somat Cell Mol Genet 1989 Sep
PMID:Isolation and characterization of S49 mouse lymphoma cell mutants deficient in adenosine deaminase. 278 37


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