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)

Lipolysis in rat adipocytes is controlled by the hormonally mediated stimulation and inhibition of adenylate cyclase. This dual regulation involves stimulatory (Gs) and inhibitory (Gi) GTP-binding proteins which control cAMP production in a GTP dependent manner. Adenosine, acting via the A1 receptor-Gi complex provides tonic regulation of adenylate cyclase and lipolysis in rat adipocytes. Adipocytes prepared from young obese Zucker (fa/fa) rats exhibit less stimulation (or greater inhibition) in response to adenosine deaminase, alone or in combination with lipolytic hormones, as compared with their lean littermates. Adenylate cyclase, measured in membranes prepared from obese adipocytes, showed decreased sensitivity to activation by low concentrations of GTP and was not inhibited by higher concentrations of the guanine nucleotide which, in lean control rats results in a biphasic activity curve. Adenosine A1 receptor binding, measured in these same membranes, demonstrated an increased sensitivity to activation by the GTP analogue, guanylyl imidodiphosphate. The presence of the analogue results in the dissociation of the receptor-Gi complex and conversion to the low affinity form in a greater proportion of receptors in the obese membranes. These results are consistent with an increased sensitivity to adenosine mediated inhibition of adenylate cyclase and lipolysis in the fat cells of the young obese (fa/fa) Zucker rat.
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PMID:Winner of the 1988 NAASO Young Investigator Award. Regulation of fat cell adenylate cyclase in young Zucker (fa/fa) rats: alterations in GTP sensitivity of adenosine A1 mediated inhibition. 196 33

Adenosine, acting at A1 and A2 purine nucleoside receptors, regulates the physiology of many tissues. Myometrial smooth muscle from pregnant guinea pigs, which is contracted by the actions of adenosine, possesses an A1 receptor whose agonist affinity is regulated by guanine nucleotides. In addition to its expected effect on the affinity of the A1 receptor for agonist, the addition of guanine nucleotide also dramatically increases antagonist binding by as much as 62%. This action of guanine nucleotides on adenosine A1 receptors is common to many smooth muscle preparations and suggests the possibility that GTP-binding proteins might alter the conformation of the adenosine receptor in such a way that receptors not previously able to bind ligands are recruited by the guanine nucleotide. Such an action of guanine nucleotides would alter our general view of the interaction of antagonists with GTP-binding protein coupled receptors, as well as bear significantly on the interpretation of experimental data designed to characterize purinergic receptors. Thus, we have investigated the actions of guanosine-5'-O-[3-thiotriphosphate] on A1 adenosine receptor binding in membranes prepared from pregnant guinea pig myometrium containing 61% right-side-out vesicles. We show that guanosine-5'-O-[3-thiotriphosphate] lowers the affinity of adenosine A1 receptors for agonist in vesicles leading to increased competition of antagonist radioligand for receptor. We suggest that the endogenous adenosine we measure originates from breakdown of significant amounts of adenine nucleotides present in membranes vesicles. Furthermore, we demonstrate that opening membrane vesicles to remove trapped adenosine yields maximal antagonist radioligand binding without subsequent effects of guanosine-5'-O-[3-thiotriphosphate]. We conclude that the presence of endogenous adenosine, unavailable to the actions of adenosine deaminase, is responsible for the effect of guanine nucleotides to increase antagonist binding to adenosine A1 receptors.
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PMID:On the ability of endogenous adenosine to regulate purine nucleoside receptor binding of antagonists in smooth muscle membranes. 212 9

The effect of increasing doses of GTP on agonist and antagonist binding to adenosine A1-receptors in different regions of rat brain was studied by autoradiography. A high concentration of GTP (100 microM) practically eliminated the binding of the agonist [3H]N6-cyclohexyladenosine in all regions. However, there were regional differences in the effects of low concentrations of GTP (0.1-10 microM). In some regions, for example the hippocampus, all concentrations of GTP decreased [3H]N6-cyclohexyladenosine binding, by decreasing the Bmax. In other structures, e.g. the superior colliculus, there was a biphasic response to GTP. Concentrations of 0.1-3 microM increased agonist binding, apparently due to a decrease in KD, whereas higher concentrations also decreased binding in these regions. The effects of GTP were mimicked by the stable GTP analogue guanosine-5'-O-(3-thiotriphosphate). GTP (0.5-100 microM) increased the binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine in all regions, but most markedly in those where GTP had a biphasic effect on agonist binding. Decreasing the levels of endogenous adenosine by increasing the concentration of adenosine deaminase and adding the 5'-nucleotidase inhibitor alpha-beta-methylene adenosine-5'-diphosphate gave an increase in [3H]8-cyclopentyl-1,3-dipropylxanthine binding and diminished the response to GTP. In sections treated with adenosine deaminase and alpha-beta-methylene adenosine-5'-diphosphate, GTP steadily decreased [3H]N6-cyclohexyladenosine binding in all regions. Thus, the GTP-induced increase in both agonist and antagonist binding may be due to a displacement of endogenous adenosine. In the presence of 1 mM EDTA, GTP had a monophasic effect on the binding of [3H]N6-cyclohexyladenosine in all regions. In the presence of 2 mM MgCl2 a biphasic response to GTP was seen in all regions. In EDTA washed sections, the effect of MgCl2 on [3H]N6-cyclohexyladenosine binding was more pronounced in the superior colliculus, where we had observed a biphasic response to GTP. The results suggest that there are regional differences in the effects of GTP on adenosine A1-receptor binding in rat brain, that reflect regional differences in the magnesium-dependent binding of endogenous adenosine, which is bound to the receptor by tight binding, is very difficult to remove, and easily interferes with radioligand binding in in vitro experiments. There may be regional differences in the sensitivity of A1-receptor-G-protein complexes to magnesium, that reflect a heterogeneity of the G-proteins to which the A1-receptors are coupled.
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PMID:Regional differences in the effect of guanine nucleotides on agonist and antagonist binding to adenosine A1-receptors in rat brain, as revealed by autoradiography. 235 51

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 simple and fast ion pair reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of ATP, ADP, AMP, GTP, GDP, IMP, NADP+, NADPH+, NAD+, NADH, ADP-ribose, inosine, adenosine, hypoxanthine, and xanthine. This method allows us to have a complete picture of the most important nucleotides present in fresh human erythrocytes. Furthermore it is particularly useful in the study of the erythrocyte adenine nucleotide catabolism allowing the detection of degradation products such as IMP, inosine, adenosine, hypoxanthine, and xanthine. The separation of the compounds under investigation is achieved in less than 15 min using a reversed-phase 3-micron Supelcosil LC-18 column and adding tetrabutylammonium, as ion-pair agent, to the buffers. The short time of analysis, the high reproducibility of the system, and the accurate evaluation of the compounds of interest make this method particularly suitable for routine analysis. Finally it is possible to use this assay as an alternative method of measuring activities of enzymes which catalyze reactions involving some of these compounds, as in the case of Na+-K+ ATPase, AMP deaminase, and adenosine deaminase.
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PMID:A very fast ion-pair reversed-phase HPLC method for the separation of the most significant nucleotides and their degradation products in human red blood cells. 282 56

Adenosine attenuates beta-adrenergic receptor mediated activation of adenylate cyclase in myocardial membranes via adenosine Ri receptors. The effects of adenosine analogs on the binding characteristics of beta-adrenergic receptors were examined in the present study utilizing rat ventricular membranes treated with adenosine deaminase. In 125I-cyanopindolol/isoproterenol competitive binding experiments phenylisopropyladenosine (PIA) significantly increased the IC50 for isoproterenol from 48 +/- 6 nM to 140 +/- 48 nM and steepened the slope of the competition curves from -0.56 +/- 0.03 to -0.90 +/- 0.21. Computer analysis of these curves indicated that binding of isoproterenol to the high affinity state of the beta-adrenergic receptor was eliminated in the presence of PIA. PIA had no effects in the presence of GPP(NH)P. 2-chloroadenosine, a less specific Ri agonist, caused smaller increases in IC50 and slope, without significantly affecting high affinity binding. 2',5'-dideoxyadenosine, a P-site agonist, had no significant effects on isoproterenol binding. During the time course of the competitive binding experiments the membranes displayed isoproterenol-sensitive adenylate cyclase activity in the absence of added GTP. These data suggest that adenosine attenuates catecholamine-induced activation of adenylate cyclase via Ri receptors by decreasing the ability of beta-adrenergic agonists to promote the formation of a high affinity complex composed of the agonist, receptor and stimulatory guanine nucleotide binding protein.
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PMID:The adenosine Ri agonist, phenylisopropyladenosine, reduces high affinity isoproterenol binding to the beta-adrenergic receptor of rat myocardial membranes. 284 21

Of the various species of cellular 5'-nucleotidases, membranous, lysosomal and cytosolic, only the latter are likely to play a role in the physiologic dephosphorylation of the 5'-nucleoside monophosphates present in the cytoplasm. The necessity to preserve cellular ATP renders a strict control of the dephosphorylation as well as of the deamination of AMP mandatory, because both nucleotides are maintained in equilibrium by adenylate kinase. Our studies of cytosolic purine 5'-nucleotidases purified from rat liver and from human erythrocytes, reviewed in this presentation, have shown that both display complex kinetic properties. Both enzymes have markedly higher affinities for IMP and for GMP than for AMP. In addition, they are stimulated by nucleoside triphosphates, among them ATP and GTP, and inhibited by Pi. The erythrocytic purine 5'-nucleotidase is also stimulated by glycerate 2,3-bisphosphate. It could thus be expected that under conditions of ATP and GTP breakdown, particularly when accompanied by an increase in Pi, the dephosphorylation of AMP would be curtailed. To verify this hypothesis, experiments were performed with isolated rat hepatocytes and with human red blood cells. The rate of dephosphorylation of AMP was measured by following time-wise the production of adenosine in the presence of coformycin (or deoxycoformycin) and 5-iodotubercidin. The coformycins inhibit the deamination of adenosine into inosine by adenosine deaminase, and 5-iodotubercidin inhibits the recycling of adenosine into AMP by adenosine kinase. Upon induction of ATP catabolism by the addition of fructose to isolated rat hepatocytes, the dephosphorylation of AMP was nearly completely suppressed. In accordance with these results, the activity of the rat liver cytosolic 5'-nucleotidase, assayed in the presence of concentrations of substrate and effectors mimicking those measured in intact cells following the addition of fructose, was decreased as compared to control conditions. In hepatocytes in which ATP catabolism was induced by suppression of oxygen, the rate of dephosphorylation of AMP increased about 3-fold. However, in contradiction with these data, the activity of the cytosolic 5'-nucleotidase, measured under conditions mimicking anoxia, decreased markedly. In human erythrocytes, dephosphorylation of AMP did not occur under physiologic conditions, but proceeded when ATP catabolism was induced by glucose lack or by alkalinization. The rate of dephosphorylation of AMP was 3-fold higher during glucose deprivation than under alkaline conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cytosolic purine 5'-nucleotidases of rat liver and human red blood cells: regulatory properties and role in AMP dephosphorylation. 285 49

Theophylline inhibits basal adenylate cyclase activity as well as cyclase stimulated by sodium chloride, sodium fluoride, GTP or 5'-guanylimidodiphosphate. This inhibition, is dose-dependent and shows non-competitive inhibition, with respect to MgATP. The presence of adenosine deaminase does not alter the effect of theophylline. The inhibition produced by theophylline is not additive with that due to 2'-deoxyadenosine 3'-monophosphate (a P-site agonist). It is suggested that theophylline may act at the P-site to reduce adenylate cyclase activity.
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PMID:Inhibition of rabbit cardiac adenylate cyclase by theophylline. 286 8

Despite numerous reports of solubilization of adenosine A1 receptors, little progress has been made in isolating or purifying the receptor, owing to the extreme lability of the preparations. The present solubilization strategies recognized the possible role of endogenous adenosine to produce adenosine-receptor-N-protein complexes, which are intrinsically unstable, and instead attempted to use caffeine to solubilize free adenosine receptors, which might be more stable. Endogenous adenosine was removed from membranes by using adenosine deaminase along with GTP to accelerate the release of receptor-bound adenosine. The receptors were then occupied with caffeine and solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) in the presence of glycerol. These soluble preparations exhibited the characteristics of free adenosine receptors. They bound the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPDPX) with high affinity to a single class of binding sites, which were insensitive to GTP. The binding activity was extremely stable, with a half-life of about 5 days at 4 degrees C; there was little change in either receptor number or affinity during 3 days at 4 degrees C. This methodology should greatly facilitate the characterization, isolation and purification of the adenosine A1 receptor.
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PMID:Solubilization of stable adenosine A1 receptors from rat brain. 293 Apr 58

Binding of (-)N6-phenylisopropyl[3H]adenosine ([3H]PIA) to intact rat fat cells was studied in the presence of the adenosine uptake blocker dipyridamole. Specific binding of 5 nmol/l [3H]PIA at 37 degrees C was rapid, reversible and dependent on cell concentration and the presence of adenosine deaminase. Saturability of specific binding was not achieved at concentrations up to 200 mumol/l [3H]PIA. In competition experiments (-)PIA (IC50 42 nmol/l) was the most potent analogue, followed by 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine. Binding of [3H]PIA was stereospecific, since (-)PIA was 200 times more potent than (+)PIA. The adenosine antagonist theophylline inhibited binding with an IC50 of 16.9 mumol/l, whereas adenine, inosine and GTP did not affect binding. The results allow us to describe several characteristics of [3H]PIA binding to intact fat cells but a considerable component of nonreceptor binding impedes a detailed study of adenosine receptors under physiological conditions.
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PMID:Study of adenosine receptors in intact rat fat cells by radioligand binding. 298 59


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