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)

The present work describes an assay which is highly specific for ribose-5-phosphate. The method is based on the following three-stage enzymatic conversion: (1) ribose 5-phosphate in equilibrium ribose 1-phosphate (phosphopentomutase); (2) ribose 1-phosphate + adenine in equilibrium adenosine + Pi (adenosine phosphorylase); (3) adenosine + H2O----inosine + NH3 (adenosine deaminase). Ribose 5-phosphate may be determined either directly following the change in absorbance at 265 nm associated with the conversion of adenine to inosine, or radioenzymatically by measuring the radioactivity of inosine formed from [8-14C]adenine, after chromatographic separation of the nucleoside on polyethyleneimine-cellulose. The spectrophotometric assay was used to follow ribose 5-phosphate formation and ribose 1-phosphate consumption catalyzed by phosphopentomutase. Further, the ability of alkaline phosphatase, 5'-nucleotidase and crude extract of Bacillus cereus cells to act on ribose 5-phosphate was tested. The radioenzymatic assay was proved useful in determining the levels of ribose 5-phosphate in rat tissues.
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PMID:Spectrophotometric and radioenzymatic determination of ribose-5-phosphate. 653 May 7

Hot water extracts of Mo-er (1 gm by 15 ml of water), an oriental food (Auricularia auricula), inhibit strongly both human and rat platelet ADP-induced aggregation. HPLC analysis of two varieties of Mo-er, A. auricula and A. polytricha (a black tree fungus), shows that they contain adenosine (Ado), 133 and 154 micrograms per gram of dry fungus, respectively. The inhibition of ADP-induced platelet aggregation by Mo-er extracts and by Ado was compared. Mo-er extracts caused a more rapid onset and a longer duration of inhibition that produced by equivalent amounts of Ado. Furthermore, Mo-er extract treated with adenosine deaminase to degrade the Ado retained the capacity to inhibit platelet aggregation. The inhibitory effects of Mo-er extracts of ADP-induced human platelet aggregation are greatly potentiated by the inhibitors of cyclic AMP phosphodiesterase such as oxagrelate (phthalazinol) and papaverine. The inhibition of platelet aggregation is only partially blocked by 2',5'-dideoxy-adenosine (DDA), an inhibitor of platelet adenylate cyclase and 5'-deoxy, 5'-methylthioadenosine (MTA), an antagonist of ADO receptors. ADP-induced rat platelet aggregation is strongly inhibited by Mo-er extracts, but not by Ado. This inhibition is not reversed by either DDA or MTA. These findings indicate that Mo-er extracts contain an agent (or agents) in addition to Ado, that blocks platelet aggregation by a mechanism that does not involve the platelet cyclic AMP system.
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PMID:Inhibition of human and rat platelet aggregation by extracts of Mo-er (Auricularia auricula). 698 40

The synthesis of the fluorescent derivative of adenosine, by reaction with 5-(dimethylamino)naphthalene-1-sulfonyl chloride in dry pyridine at low temperature, yielding 3'-O-[5-(dimethylamino)naphthalene-1-sulfonyl]adenosine (3'-O-dansyladenosine), is here described. 3'-O-Dansyladenosine is partially soluble in water (approximately 10(-4) M) and upon excitation at 325 nm exhibits maximum fluorescence emission at 516 +/- 22 nm (corrected) in buffered aqueous solution at pH 7.6 with a quantum yield of 0.21 and a lifetime of 11.8 +/- 0.2 ns. The fluorescence of 3'-O-dansyladenosine is sensitive to the polarity of its solvent: in pyridine, a quantum yield of 0.61 at the emission maximum of 435 nm was observed. 3'-O-Dansyladenosine is a reversible competitive inhibitor of adenosine deaminase with a moderate inhibitive dissociation constant K1 = (1.54 +/- 0.13) X 10(-5) M. The enzyme-substrate analogue association constant was determined by equilibrium dialysis to be K = (0.69 +/- 0.05) X 10(5) M-1, very close to KI-1. The hydrophobic nature of its binding site in adenosine deaminase is evident from the strong blue shift of the fluorescence emission maximum to 440 nm, the 4-fold increase in fluorescence quantum yield, and the longer lifetime of 15.8 +/- 0.2 ns; the tight, rigid nature of the complex is evident from its high fluorescence polarization value, 0.23. An 85% decrease in the fluorescence emission intensity of the adenosine deaminase-3'-O-dansyladenosine complex in the presence of adenosine indicates the selective binding to the enzyme active site. Correlation between the conformation of the probe, either when free in various solvents or when bound to the enzyme, and its fluorescence quantum yield is noted. 3'-O-Dansyladenosine is suitable for fluorescent labeling of adenosine deaminase in cell systems.
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PMID:Fluorescent substrate analogue for adenosine deaminase: 3'-O-[5-(dimethylamino)naphthalene-1-sulfonyl]adenosine. 724 72

A radioisotopic method for a rapid assay of adenosine deaminase in human lymphocytes is proposed here. [2-3H] adenosine, as substrate, has been employed for the enzymatic assay. The products of reaction have been resolved by thin layer chromatography on PEI cellulose. The plates were developed with distilled water and inosine spots absorbing in the U.V. were eluted with 0.1 N HCl. The eluates obtained from the inosine spots were employed for radioactive measurements.
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PMID:Radioisotopic microassay of adenosine deaminase in human lymphocytes. 727 86

A new spectrophotometric method for the determination of adenosine deaminase is described. Adenosine is deaminated to inosine, the latter is cleaved by an inosine-guanosine specific nucleoside phosphorylase to hypoxanthine and ribose-1-phosphate. Hypoxanthine can be oxidized further to uric acid by xanthine oxidase or to allantoin by xanthine oxidase and uricase. The hydrogen peroxide formed in these reactions is reduced by catalase to water. In the presence of high concentrations of ethanol, equivalent amounts of acetaldehyde are produced. The acetaldehyde is oxidized NAD(P) dependent and the production rate of NAD(P)H is recorded at 334 nm. The new method is suitable for the detection of adenosine deaminase in whole blood, lymphocytes, sera and tissues.
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PMID:A new spectrophotometric assay for enzymes of purine metabolism. IV. Determination of adenosine deaminase. 736 76

Three new missense mutations (H15D, A83D, and A179D) and a new splicing defect (573 + IG-->A) in the 5' splice site of intron 5 were among six mutant adenosine deaminase (ADA) alleles found in three unrelated patients with severe combined immunodeficiency disease, the most common phenotype associated with ADA deficiency. When expressed in vitro, the H15D, A83D, and A179D proteins lacked detectable ADA activity. The splicing defect caused skipping of exon 5, resulting in premature termination of translation and a reduced level of mRNA. H15D is the first naturally occurring mutation of a residue that coordinates directly with the enzyme-associated zinc ion. Molecular modeling based on the atomic coordinates of murine ADA suggests that the D15 mutation would create a cavity or gap between the zinc ion and the side chain carboxylate of D15. This could alter the ability of zinc to activate a water molecule postulated to play a role in the catalytic mechanism. A83 and A179 are not directly involved in the active site, but are conserved residues located respectively in alpha helix 4 and beta strand 4 of the alpha/beta barrel. Replacement of these small hydrophobic Ala residues with the charged, more bulky Asp side chain may distort ADA structure and affect enzyme stability or folding.
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PMID:Four new adenosine deaminase mutations, altering a zinc-binding histidine, two conserved alanines, and a 5' splice site. 759 35

Earlier studies have shown guanine arabinoside (ara-G) is an effective agent against growth of T-cell lines and freshly isolated human T-leukemic cells. However, poor water solubility of ara-G limits clinical use. 2-Amino-6-methoxypurine arabinoside (506U) is a water-soluble prodrug converted to ara-G by adenosine deaminase. 506U is not a substrate for deoxycytidine kinase, adenosine kinase, or purine nucleoside phosphorylase and is phosphorylated by mitochondrial deoxyguanosine kinase at a rate 4% that of ara-G phosphorylation. Mitochondrial DNA polymerase was the least sensitive to ara-GTP inhibition of the five human DNA polymerases tested. [3H]506U was anabolized to ara-G 5'-phosphates in CEM cells but not to phosphorylated metabolites of 506U. 506U was selective for transformed T over B cells and also inhibited growth in two of three monocytic lines tested. 506U given i.v. to cynomolgus monkeys was rapidly converted to ara-G; the ara-G had a half-life of approximately 2 h. 506U had in vivo dose-dependent efficacy against human T-cell tumors in immunodeficient mice. A Phase 1 trial of 506U against refractory hematological malignancies is now in progress at two study sites.
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PMID:2-Amino-6-methoxypurine arabinoside: an agent for T-cell malignancies. 761 70

A mechanism which mediates the transport of the nonphysiological nucleoside, L-adenosine, was demonstrated in Plasmodium falciparum infected erythrocytes and naturally released merozoites. L-Adenosine was not a substrate for influx in freed intraerythrocytic parasites or in normal human erythrocytes nor was L-adenosine transported in a variety of cell types including other parasitic protozoa such as Crithidia luciliae, Trichomonas vaginalis, Giardia intestinalis, or the mammalian cells, Buffalo Green Monkey and HeLa cells. L-Adenosine transport in P. falciparum infected cells was nonsaturable, with a rate of 0.13 +/- 0.01 pmol/microliter cell water per s per microM L-adenosine, yet the transport was inhibited by furosemide, phloridzin and piperine with IC50 values between 1-13 microM, distinguishing the transport pathway from simple diffusion. The channel-like permeation was selective as disaccharides were not permeable to parasitised cells. In addition, an unusual metabolic property of parasitic adenosine deaminase was found in that L-adenosine was metabolised to L-inosine by both P. falciparum infected erythrocytes and merozoites, an activity which was inhibited by 50 nM deoxycoformycin. No other cell type examined displayed this enzymic activity. The results further substantiate that nucleoside transport in P. falciparum infected cells was significantly altered compared to uninfected erythrocytes and that L-adenosine transport and metabolism was a biochemical property of Plasmodium infected cells and merozoites and not found in normal erythrocytes nor any of the other cell types investigated.
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PMID:Parasite-induced permeation of nucleosides in Plasmodium falciparum malaria. 779 64

Purified striatal synaptosomes were superfused continuously with L-[3,5-3H]tyrosine to measure simultaneously the synthesis ([3H]water formed during the conversion of [3H]tyrosine into [3H]DOPA) and the release of [3H]dopamine ([3H]DA). Glutamate (10(-3) M) and NMDA (10(-3) M, in the absence of Mg2+) stimulated the release of [3H]DA, but they reduced the efflux of [3H]water. This reduction of [3H]DA synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Although D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate stimulated the release of [3H]DA, they did not affect its synthesis. The glutamate-evoked inhibition of [3H]DA synthesis was prevented when synaptosomes were superfused continuously with adenosine deaminase plus quinpirole, a treatment which markedly reduces the phosphorylation of tyrosine hydroxylase by cAMP dependent protein kinase. The opposite effects of glutamate on [3H]DA synthesis and release were mimicked by ionomycin (10(-6) M). It is proposed that both an activation of a cyclic nucleotide phosphodiesterase and a dephosphorylation of tyrosine hydroxylase linked to the influx of calcium through NMDA receptors is responsible for the inhibition of dopamine synthesis by glutamate and that calcineurin could play a critical role in these processes.
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PMID:Opposite presynaptic regulations by glutamate through NMDA receptors of dopamine synthesis and release in rat striatal synaptosomes. 791 26

Purified striatal synaptosomes were continuously superfused with L,3,5[3H]tyrosine in order to estimate the synthesis ([3H]water) and release of newly formed [3H]dopamine. In the presence of magnesium, L-glutamate, D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate, but not N-methyl-D-aspartate (NMDA) and 1-aminocyclopentane-1S,3R-dicarboxylate (t-ACPD), stimulated the release of [3H]dopamine, in a dose-dependent manner. When magnesium was omitted or in the presence of AMPA, NMDA also increased the release of [3H]dopamine. The effects of AMPA and kainate were competitively inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 6,7-dinitro-quinoxaline-2,3-dione (DNQX), whereas those of NMDA were reduced by 2-amino-5-phosphonovalerate (APV) or (+)-5-methyl-10,11-dihydro-5-H-dibenzo(a,d)cyclo-hepten-5,10-imine maleate (MK801). The stimulation of [3H]dopamine release by a high concentration of glutamate resulted from the concomitant activation of AMPA and NMDA receptors since this effect was potentiated by glycine and reduced by 2-amino-5-phosphonovalerate or MK801. This reduction was almost complete in the combined presence of DNQX and MK801. Surprisingly, glutamate and NMDA (in the absence of magnesium) reduced the efflux of [3H]water. The reduction of [3H]dopamine synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Neither AMPA nor kainate affected dopamine synthesis. The inhibition of [3H]dopamine synthesis resulting from the stimulation of NMDA receptors was prevented when synaptosomes were continuously superfused with adenosine deaminase and quinpirole, a combined treatment known to markedly reduce the phosphorylation of tyrosine hydroxylase by cAMP-dependent protein kinase. The opposite effects of a high concentration of glutamate on [3H]dopamine synthesis and release were mimicked by ionomycin. As a working hypothesis, it is proposed that the NMDA-triggered calcium influx could lead to a reduction of tyrosine hydroxylase phosphorylation, possibly through an activation of calcineurin.
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PMID:Presynaptic control of dopamine synthesis and release by excitatory amino acids in rat striatal synaptosomes. 799 95


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