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)

Gelfiltered platelets (GFP) in calcium free Tyrode solution containing albumin, glucose and adenosine deaminase were preincubated with 1 micronM 14C-ADP or 0.15 M NaCl (control) at 37 degrees C. The breakdown of extracellular 14C-ADP was markedly inhibited in this medium. No aggregation took place without fibrinogen, but the platelets underwent a disc to sphere transformation with development of refactoriness towards ADP. Presence of 2 mM CaCl2 in the incubation medium did not prevent refractoriness as reported earlier with washed rabbit platelets. When the ADP degrading enzyme, apyrase, was added at 30 min of incubation a partial recovery of the aggregability was observed. Electron microscopic studies showed that the partial restoration of the aggregation response, due to ADP degradation by apyrase, was accompanied by a return of discoidal morphology of the platelets. The ultrastructural studies showed further that spherical form with large number of pseudopods is not by itself a necessary or sufficient indication of platelets in a refractory state. However, the results indicated that spherical platelets are more vulnerable to external factors. It was concluded that refractoriness was mainly caused by a direct effect on the platelets by ADP itself, but the studies also suggested that deteriorating, irreversible, intracellular changes may take place when platelets are in spherical shape. An artificial medium, mechanical stress, incubation at 37 degrees C are factors that probably speed up these changes.
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PMID:ADP-induced refractory state of platelets in vitro. II. Functional and ultra studies on gel filtered platelets. 85 91

The role of platelets in the maintenance of endothelial barrier is examined in an in vitro model of the microvasculature. Human platelets (6,000/microliters) perfused through a cell column of endothelial-covered microcarriers decrease paracellular permeability of sodium fluorescein (mol wt 342) to 63% of baseline values. This effect is reversible and a second application and removal of platelets produces a similar response. This effect occurs within 5 min and reverses within 10 min after platelet removal. The reduction in permeability is not due to mechanical obstruction of endothelial junctions, since the number of recirculating platelets is not reduced and releasate from unstimulated 2-h platelet incubations also decreases permeability. Releasate from platelets stimulated with 0.1 U/ml of thrombin for 15 min have the same permeability reducing effect. In this system, the platelet factors serotonin (10(-3) M) and ADP (10(-4) M) have no effect on permeability. However, the platelet factors adenosine (10(-4) M), ATP (10(-5) M), and beta-agonists decrease permeability. None of these appear to account for platelet permeability activity, since activity is not blocked by agents directed against these mediators (adenosine deaminase, apyrase, 8-phenyltheophylline, or propranolol). The active factor(s) is stable at -20 degrees C, heat stable, sensitive to trypsin, and has an apparent molecular weight > 100. We conclude that unstimulated platelets release a factor(s) that enhances endothelial barrier in vitro and may be important in maintenance of the normal in vivo barrier.
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PMID:Platelets and a platelet-released factor enhance endothelial barrier. 147 5

cAMP is commonly measured using either immunoassay or high-performance liquid chromatography. The current methods are sensitive but may lack versatility and be expensive; also, radioactivity is potentially harmful to the operator and environment. Given these concerns, we developed a highly sensitive enzymatic fluorometric assay for cAMP. The method consists of five steps: (1) destruction of interfering compounds with apyrase, 5' nucleotidase, adenosine deaminase, and alkaline phosphatase; (2) conversion of cAMP to AMP; (3) conversion of AMP to ATP; (4) amplification of ATP by ATP-ADP cycling; and (5) fluorometric measurement of resultant NADPH. cAMP was measured in male Sprague Dawley rats anesthetized with pentobarbital. Stimulated rats (n = 4) received isoproterenol (16 micrograms/kg, s.q.) and aminophylline (20 mg/kg, s.q.), whereas controls (n = 4) received no additional drug. With the enzymatic fluorometric assay, cAMP content in heart, liver, and kidney (pmol/mg wet wt, mean +/- SEM) was 0.34 +/- 0.03, 0.33 +/- 0.03, and 0.92 +/- 0.11 in the control group and 0.77 +/- 0.10, 0.66 +/- 0.04, and 1.53 +/- 0.12 in the stimulated group, respectively. The total assay duration including sample reading procedure varied at 4.5-9.5 hr, depending on its sensitivity. cAMP from the same samples was measured using a commercially available enzyme immunoassay kit and was found to be very similar to the enzymatic fluorometric assay. We conclude that this new assay is sensitive, safe, versatile, and inexpensive and can be used to measure cAMP in multiple types of tissue, including biopsy samples weighing < 200 micrograms.
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PMID:Enzymatic fluorometric assay for tissue cAMP. 786 85

The effect of human platelets on chemoattractant-induced generation of oxygen metabolites in neutrophils was investigated, using luminol-enhanced chemiluminescence (CL). Resting platelets inhibited the extracellular, but not the intracellular, production of oxygen radicals in formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils. Maximal effect was obtained at the physiological neutrophil/platelet ratio of 1/50. Similar results were acquired by adding supernatants of platelets, indicating a role for a soluble factor. Removal of extracellular adenosine by adenosine deaminase (ADA), or blocking of adenosine-receptors by theophylline, antagonized the inhibitory effects of platelets (or the equivalent supernatant) on the neutrophil respiratory burst. In contrast, accumulation of adenosine by apyrase enhanced the inhibition. Exogenous adenosine mimicked the effects of platelets on the fMet-Leu-Phe-induced respiratory burst. To further assess the role of platelet-derived adenosine, the platelets were fixed with paraformaldehyde. We found that fixed platelets, as well as their supernatant, inhibited the fMet-Leu-Phe-induced CL-response to the same extent as viable cells. These effects were also reversed by ADA and theophylline, respectively. A prior removal of adenosine in the platelet suspension by ADA, followed by treatment with erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) to inactivate ADA, did not reverse the inhibitory action of platelets on the fMet-Leu-Phe-induced CL-response in neutrophils. However, if adenosine receptors of neutrophil at the same time were blocked with theophyline, the inhibition was significantly reduced. Platelets markedly increased the generation of adenosine in a neutrophil suspension. The effect was antagonized by S-(4-Nitrobenzyl)-6-thioguanosine (NBTG), but unaffected by alpha, beta-methyl-eneadenosine5'diphosphate (AMP-CP), indicating that the platelet-dependent accumulation of adenosine is due to an increased release of endogenous adenosine from neutrophils and not to a degradation of extracellular AMP. In correlation, NBTG, but not AMP-CP, reversed the platelet-mediated inhibition of the fMet-Leu-Phe-induced CL-response in neutrophils. Consequently, these data suggest that a platelet-derived factor increases the release of endogenously formed adenosine from neutrophils, terminating the production of oxygen radicals. The inhibition of oxidase activity was also associated with a platelet-induced polymerization of actin in the margin of the neutrophils. Treatment of neutrophils with cytochalasin B reversed the effects of platelets, both on F-actin content and CL-response. In summary, resting platelets limit the release of oxygen radicals from chemoattractant-stimulated neutrophils, thus preventing excessive damage to host tissues in the vascular space. This effect is suggested to be associated with an increase generation of neutrophil-derived adenosine enhancing an autoregulatory inhibitory pathway, and a peripheral accumulation of actin filaments forming a barrier for extracellular release of reactive oxygen radicals.
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PMID:Release of oxygen metabolites from chemoattractant-stimulated neutrophils is inhibited by resting platelets: role of extracellular adenosine and actin polymerization. 863 3

The interaction of platelets with neutrophil granulocytes is considered to play an important role in the inflammatory process, and the present study was focused on platelet-induced modulation of Fcgamma receptor-mediated functions in neutrophils. We found that phagocytosis and the respiratory burst (measured as luminol-enhanced chemiluminescence), triggered in neutrophils by immunoglobulin G (IgG)-opsonized yeast particles, were potentiated by platelets and that maximal enhancement was achieved at a physiological neutrophil/platelet ratio of about 1:50 to 1:100. Platelets both increased the intra- and extracellular generation of oxygen radicals as well as the release of myeloperoxidase from stimulated neutrophils. The presence of platelets also induced a cortical actin polymerization in neutrophils, which might explain the increased phagocytic capacity. Platelets appear to affect neutrophil function in a contact-independent manner that most likely involves ATP, indicated by the following: (1) platelet supernatants, but not fixed platelets, affected neutrophil function in the same way as viable platelets; (2) platelets raised the extracellular ATP level four- to fivefold; (3) exogenous ATP mimicked the effects of platelets on actin polymerization, phagocytosis, and the respiratory burst in neutrophils; (4) hydrolysis of extracellular ATP with apyrase or blocking of ATP receptors with suramin reversed the platelet-induced enhancement of neutrophil function. An increased accumulation of extracellular adenosine, induced by inhibiting endogenous adenosine deaminase or adding exogenous adenosine, reversed the effects of platelets. The platelet-induced potentiation of the respiratory burst was inhibited by the tyrosine kinase inhibitor genistein, suggesting that tyrosine phosphorylation is involved. However, platelets did not significantly affect the Fcgamma receptor-triggered calcium response in neutrophils. In conclusion, we show that platelets, through an ATP-dependent mechanism, potentiate IgG-mediated ingestion and production of oxygen metabolites in neutrophils.
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PMID:Platelets enhance Fc(gamma) receptor-mediated phagocytosis and respiratory burst in neutrophils: the role of purinergic modulation and actin polymerization. 869 24

In the present investigation, effect of rat peripheral polymorphonuclear leukocyte (PMNL) supernatant was investigated on platelet aggregation. Rat PMNLs suspended in Hanks balanced salt solution (HBSS, pH 7.4) were incubated at 37 degrees C for different time intervals and cell-free supernatant was obtained by centrifugation. Supernatant was found to inhibit adenosine diphosphate (ADP), arachidonic acid (AA), and calcium ionophore-induced platelet aggregation. The inhibitory effect of PMNL supernatant on platelet aggregation was not blocked by methylene blue (10 microM) or adenosine deaminase (5 U ml(-1)) pretreatment, suggesting that the inhibitory effect of the supernatant on aggregation was not mediated by nitric oxide (NO) or ecto-ADPase. The effect of PMNL supernatant on platelet aggregation was abolished by preheating the supernatant at 95 degrees C for 5 minutes. Pretreatment of the supernatant with protease inhibitors abolished the inhibitory effect of supernatant on platelet aggregation suggesting that the factor may be a protein or peptide with protease activity. Partial purification of biologically active factor by fine particle liquid chromatography (FPLC) by using Superose 6B column yielded a peak with a molecular weight of approximately 30 kDa having antiaggregatory activity. The results obtained suggest that rat peripheral PMNLs release yet another factor(s) that inhibits platelet aggregation. The factor is a heat labile protein with a molecular weight of approximately 30 kDa.
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PMID:Inhibition of platelet aggregation by a protein factor present in rat peripheral polymorphonuclear leukocyte supernatant. 972 23

Cyclic adenosine diphosphate ribose and adenosine diphosphate ribose (ADPR) play an important role in the regulation of intracellular Ca(2+) release and K(+) channel activity in the coronary arterial smooth muscle. The role of these signaling nucleotides in the control of vascular tone has yet to be determined. The present study was designed to determine whether ADPR produces vasodilation in coronary arteries and to explore the mechanism of action of ADPR. ADPR (10-60 micromol/l) was found to produce endothelium-independent relaxation in a concentration-dependent manner in isolated and pressurized small bovine coronary arteries. The ADPR-induced vasodilation was substantially attenuated by adenosine deaminase (0.2 U/ml), and the P(1) purinoceptor antagonist 8-(p-sulfophenyl)theophylline (50 micromol/l), with maximal inhibitions of 60 and 80%, respectively. When the coronary arterial homogenates were incubated with ADPR, the production of adenosine and 5'-AMP was detected. The adenosine production was blocked by the 5'-nucleotidase inhibitor, alpha,beta-methylene adenosine 5'-diphosphate (MADP, 1 mmol/l), which was accompanied by a corresponding accumulation of 5'-AMP. This 5'-AMP accumulation was substantially inhibited by the apyrase inhibitor sodium azide (10 mmol/l). Moreover, ADPR was hydrolyzed into 5'-AMP by purified apyrase. In agreement with their inhibitory effect on the adenosine production, MADP and sodium azide significantly attenuated the vasodilator response to ADPR. The metabolism of ADPR to adenosine was only detected in cultured coronary arterial smooth muscle cells but not in endothelial cells. We concluded that ADPR produces vasodilation in small coronary arteries and that the action of ADPR is associated with the adenosine production via an apyrase- and 5'-nucleotidase-mediated metabolism.
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PMID:Adenosine diphosphate ribose dilates bovine coronary small arteries through apyrase- and 5'-nucleotidase-mediated metabolism. 1117 96

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 +/- 6 and 21 +/- 2 nmol Pi mg(-1) min(-1) for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 +/- 2 nmol Pi mg(-1) min(-1) for AMP and 1.52 +/- 0.13 nmol adenosine mg(-1) min(-1), respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules.
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PMID:Ectonucleotidase activities in Sertoli cells from immature rats. 1159 98

Extracellular purines are important signalling molecules in the vasculature that are regulated by a network of cell surface ectoenzymes. By using human endothelial cells and normal and leukaemic lymphocytes as enzyme sources, we identified the following purine-converting ectoenzymes: (1) ecto-nucleotidases, NTP diphosphohydrolase/CD39 (EC 3.6.1.5) and ecto-5'-nucleotidase/CD73 (EC 3.1.3.5); (2) ecto-nucleotide kinases, adenylate kinase (EC 2.7.4.3) and nucleoside diphosphate kinase (EC 2.7.4.6); (3) ecto-adenosine deaminase (EC 3.5.4.4). Evidence for this was obtained by using enzyme assays with (3)H-labelled nucleotides and adenosine as substrates, direct evaluation of gamma-phosphate transfer from [gamma-(32)P]ATP to AMP/NDP, and bioluminescent measurement of extracellular ATP synthesis. In addition, incorporation of radioactivity into an approx. 20 kDa surface protein was observed following incubation of Namalwa B cells with [gamma-(32)P]ATP. Thus two opposite, ATP-generating and ATP-consuming, pathways coexist on the cell surface, where basal ATP release, re-synthesis of high-energy phosphoryls, and selective ecto-protein phosphorylation are counteracted by stepwise nucleotide breakdown with subsequent adenosine inactivation. The comparative measurements of enzymic activities indicated the predominance of the nucleotide-inactivating pathway via ecto-nucleotidase reactions on the endothelial cells. The lymphocytes are characterized by counteracting ATP-regenerating/adenosine-eliminating phenotypes, thus allowing them to avoid the lymphotoxic effects of adenosine and maintain surrounding ATP at a steady-state level. These results are in agreement with divergent effects of ATP and adenosine on endothelial function and haemostasis, and provide a novel regulatory mechanism of local agonist availability for nucleotide- or nucleoside-selective receptors within the vasculature.
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PMID:The evidence for two opposite, ATP-generating and ATP-consuming, extracellular pathways on endothelial and lymphoid cells. 1209 90

Extracellular nucleotides are signaling molecules whose receptor-mediated effects are involved in a variety of physiological responses in mammalian tissues. An overwhelming body of data indicate that inflammatory and other immune responses can be modulated by the availability and local concentrations of nucleotides via nucleotide receptor signaling, but this is only just beginning to be investigated in the context of infectious disease. Evidence is provided here that the parasitic nematode Trichinella spiralis can catalyze the conversion and thus modulate both the availability and concentration of extracellular nucleotides by means of the following secreted exoenzymes: apyrase, 5'-nucleotidase, and adenosine deaminase. These enzymes were characterized in terms of substrate specificity, kinetic behavior, pH, divalent cation preferences, and response to a series of compounds. The secreted 5'-nucleotidase was identified as a protein with an apparent molecular mass of 67 kDa after N-terminal amino acid sequencing of the purified protein. The presence of adenosine deaminase was confirmed in the secreted products by Western blotting with an antibody against a mammalian enzyme, as a protein with an apparent molecular mass of 38 kDa. These secreted proteins constitute an enzymatic cascade which catalyzes the degradation of extracellular nucleotides, with a potential physiological role in the regulation of purinergic signaling.
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PMID:Nucleotidase cascades are catalyzed by secreted proteins of the parasitic nematode Trichinella spiralis. 1218 37


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