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Query: EC:3.1.3.5 (5'-nucleotidase)
 3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The characteristics of 5'-nucleotidase in a clonal line (C6) of rat glioma cells has been examined in detail. The cells liberated 6.80 +/- 0.33 mumol of inorganic phosphate/mg of cell protein/hour, producing nearly equimolar amounts of adenosine and inorganic phosphate from AMP in the extracellular fluid. No 5'-nucleotidase was released by the cells into the medium. Most of the 5'-nucleotidase activity was found to be located in the outer surface of the plasma membrane of C6 cells and rapidly accessible to exogenous AMP, by experiments based upon differential labeling of extracellular and intracellular compartments with 32P and 33P. The ecto-enzyme was active in the absence of divalent cations. However, Mn2+ or Co2+ were somewhat stimulatory. Zn2+ suppressed activity very markedly. The relationship of enzymatic reaction velocity to pH was complex, with an optimum at pH 7.4 for all substrates tested. The ecto-5'-nucleotidase readily hydrolyzed 5'-AMP and 5'-UMP. Other 5'-nucleoside monophosphates, including 5'-deoxy-AMP, were also hydrolyzed, but more slowly; 2'- or 3'-nucleoside monophosphates were not attacked. The ecto-5'-nucleotidase in the intact cell obeyed Michaelis-Menten kinetics. Apparent Km for AMP was 0.22 mM; apparent Km values for other substrates were similar and ranged from 0.16 to 0.18 mM. ADP exerted a very powerful inhibitory effect, behaving as a competitive inhibitor, and 5'-UMP behaved as a strictly competitive substrate for 5'-AMP. ATP and ITP were inhibitory. Of these, ITP served to increase Km for AMP. ATP did likewise, but also greatly lowered Vmax. These findings indicate that the intact cell is capable of rapid hydrolysis of exogenous 5'-AMP, to produce adenosine at the cell surface at a rate which responds directly to extracellular AMP concentration but which can be suppressed by extracellular ADP or ATP.
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PMID:Ecto-5'-nucleotidase of intact cultured C6 rat glioma cells. 81 33

The enzymatic inosine 5'-monophosphate assay described by Grassl [in, Methods of Enzymatic Analysis (H. U. Bergman, ed.), pp. 2168-2171, Academic Press, New York (1974)] is highly nonspecific, as ITP, ATP, ADP, AMP, and adenosine react stoichiometrically. The reactivity with the adenine derivatives is due to the tri- and diphosphatase activity of alkaline phosphatase (AP), coupled with adenosine deaminase (and possibly AMP deaminase) contamination of commercially available preparations of AP, purine-nucleoside phosphorylase, and/or xanthine oxidase. The inclusion of coformycin (0.05 microgram/ml), a potent inhibitor of these deaminases, completely eliminated the cross-reactivity. ITP, however, still reacted stoichiometrically due to the tri- and diphosphatase activity of AP. Meyer and Terjung [Amer. J. Physiol. 237 C111-C118 (1979)] introduced a modification of Grassl's procedure, substituting 5'-nucleotidase for AP. It has been found that this disallows reactivity with ATP, ADP, and ITP but that AMP and adenosine still react completely. Coformycin prevents this cross-reactivity. It is therefore recommended that the assay be carried out with 5'-nucleotidase (instead of AP) and coformycin, in order to achieve a more specific assay, and one more suitable for use with whole tissue extracts.
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PMID:An enzymatic inosine 5'-monophosphate assay of increased specificity. 298 81

The adenine nucleotides AMP, ADP and ATP (3 X 10(-7) M and above) inhibited contractile responses to transmural nerve stimulation in guinea-pig ileum longitudinal muscle via a prejunctional action. Nucleotides assumed to inhibit the degradation of adenine nucleotides were employed to determine whether inhibition of contractile responses was elicited by adenine nucleotides per se, or required breakdown to adenosine. The IMP or 2'-deoxy AMP enhanced the prejunctional inhibitory effect elicited by AMP. A similar enhancement of the inhibitory effect of ADP and ATP was seen after administration of IDP and ITP, respectively. The inhibitory effect of adenosine was not enhanced by inosine, IMP or IDP. The 5'-nucleotidase inhibitor, TDP enhanced inhibition elicited by ADP. In contrast, alpha, beta-meADP did not influence the prejunctional inhibitory effect elicited by the adenine nucleotides. However, the combination of alpha, beta-meADP and IMP enhanced the inhibitory effect of ATP. The postjunctional contractile effect elicited by ADP and ATP was enhanced by pretreatment with inosine nucleotides, alpha, beta-meADP or TDP, indicating decreased inactivation of ADP and ATP during concurrent nucleotide administration. The fact that the prejunctional effect of adenine nucleotides can be enhanced by forms of pretreatment known to antagonize the breakdown of adenine nucleotides, constitutes strong evidence for prejunctional action per se by adenine nucleotides.
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PMID:Neuromodulation by adenine nucleotides, as indicated by experiments with inhibitors of nucleotide inactivation. 301 Jun 39

Intact synaptosomes isolated from the electric organ of the electric ray Torpedo marmorata contain, at their surface, enzyme activities for the hydrolysis of externally applied nucleoside phosphates. The diazonium salt of sulfanilic acid, as a low-molecular-weight, slowly permeating, covalent inhibitory agent, selectively blocks these enzyme activities and leaves intracellular lactate dehydrogenase intact. The ectoenzymes comprise both a nucleoside triphosphate and diphosphate phosphohydrolase, as well as a 5'-nucleotidase. Activity of nonspecific ectophosphatases is absent. The nucleoside triphosphatase hydrolyzes almost equally well ATP, GTP, CTP, UTP, and ITP and is activated to a similar degree by Mg2+ or Ca2+. It has a high affinity for ATP (Km for ATP in the presence of Mg2+, 75 microM; in the presence of Ca2+, 66 microM). Maximal rates in the presence of Mg2+ and Ca2+ were very similar (34.8 and 32.5 nmol of Pi/min/mg of synaptosomal protein, respectively). Either Mg-ATP or Ca-ATP can act as a true substrate. ADP inhibits hydrolysis of ATP, but AMP is without effect. The nucleoside triphosphatase is not inhibited significantly by a number of inhibitors of mitochondrial Mg2+-ATPase or of Ca2+ + Mg2+-ATPases. It is, however, considerably inhibited by filipin and quercitin. The capacity of intact synaptosomes to hydrolyze also extracellular ADP, GDP, AMP, GMP, and IMP suggests that the nucleoside triphosphatase is part of an enzyme chain that causes complete hydrolysis of the respective nucleoside triphosphate to the nucleoside. We conclude that the cholinergic nerve terminals of the Torpedo electric organ can hydrolyze ATP released on coexocytosis with acetylcholine via an ectonucleoside triphosphatase activity that is different from known endogenous nerve terminal ATPases. The final product of the hydrolysis, adenosine, can then be salvaged by the nerve terminal for resynthesis of ATP. Other possible physiological functions of the ectonucleotidases are discussed.
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PMID:Ectonucleotidase activities associated with cholinergic synaptosomes isolated from Torpedo electric organ. 301 88

A membrane fraction enriched in plasma membrane marker enzymes K+-dependent p-nitrophenyl phosphatase, 5'-nucleotidase and alkaline phosphatase was prepared from rat parotid glands using Percoll self-forming gradient. This fraction contained an ATP-dependent CA2+ transport system which was distinct from those located on the endoplasmic reticulum and mitochondria of parotid glands. The Km for ATP was 0.57 +/- 0.07 mM (n = 3). Nucleotides other than ATP such as ADP, AMP, GTP, CTP, UTP or ITP were unable to support significant Ca2+ uptake. ATP-dependent Ca2+ uptake displayed sigmoidal kinetics with respect to free Ca2+ concentration with a Hill coefficient of 2.02. The K0.5 for Ca2+ was 44 +/- 3.1 nM (n = 3) and the average Vmax was 13.5 +/- 1.1 nmol/min per mg of protein. The pH optimum was 7.2. Trifluorperazine inhibited Ca2+ transport with half maximal inhibition observed at 30.8 microM. Complete inhibition was observed at 70 microM trifluorperazine. Exogenous calmodulin however had no effect on the rate of transport. Na+ and K+ ions activated Ca2+ transport at 20 to 30 mM ion concentrations. Higher concentrations of Na+ or K+ were inhibitory.
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PMID:Characterisation of an ATP-dependent Ca2+ transport system in a plasma membrane enriched fraction from rat parotid. 345 46

1. Using the incorporation of [methyl-3H]thymidine as a proliferation marker, the effects of various nucleosides and nucleotides on endothelial LLC-MK2 cells were studied. We found that ATP, ADP, AMP and adenosine in concentrations of 10 microM or higher stimulate the proliferation of these cells. 2. Inhibition of ecto-ATPase (EC 3.6.1.15), 5'-nucleotidase (EC 3.1.3.5) or alkaline phosphatase (EC 3.1.3.1) significantly diminished the stimulatory effect of ATP, indicating that the effect is primarily caused by adenosine and not by adenine nucleotides. Also, the effect depends only on extracellular nucleosides, since inhibition of nucleoside uptake by dipyridamole has no influence on proliferation. 3. Other purine nucleotides and nucleosides (ITP, GTP, inosine and guanosine) also stimulate cell proliferation, while pyrimidine nucleotides and nucleosides (CTP, UTP, cytidine and uridine) inhibit proliferation. Furthermore, the simultaneous presence of adenosine and any of the other purine nucleosides is not entirely additive in its effect on cell proliferation. At the same time any pyrimidine nucleoside, when added together with adenosine, has the same inhibitory effect as the pyrimidine nucleoside alone. 4. Apparently these proliferative effects are neither caused by any pharmacologically known P1-purinoceptor, nor are they mediated by cyclic AMP, cyclic GMP, or D-myo-inositol 1,4,5-trisphosphate as second messenger, nor by extracellular Ca2+. 5. Therefore, we conclude that various purine and pyrimidine nucleosides can influence the proliferation of LLC-MK2 cells by acting on putative purinergic and pyrimidinergic receptors not previously described.
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PMID:Regulation of proliferation of LLC-MK2 cells by nucleosides and nucleotides: the role of ecto-enzymes. 868

ATPase activity has been located on the external surface of Leishmania tropica. Since Leishmania is known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5'-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5'-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2 but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2 varying from 0.1 to 10 mM did not increase the ATPase activity. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 +/- 0.02 mM MgCl2. The apparent K(m) for Mg-ATP2- was 0.13 +/- 0.01 mM and free Mg2+ did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5 = 27.5 +/- 1.8 microM).
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PMID:Mg-dependent ecto-ATPase activity in Leishmania tropica. 914 51

The purine nucleoside cycle is a cyclic pathway composed of three cytosolic enzymes, hypoxanthine-guanine phosphoribosyltransferase, IMP-GMP specific 5'-nucleotidase, and purine-nucleoside phosphorylase. It may be considered a 'futile cycle', whose net reaction is the hydrolysis of 5-phosphoribosyl-1-pyrophosphate to inorganic pyrophosphate and ribose 1-phosphate. The availability of a highly purified preparation of cytosolic 5'-nucleotidase prompted us to reconstitute the purine nucleoside cycle. Its kinetics were strikingly similar to those observed when dialyzed extracts of rat brain were used. Thus, when the cycle is started by addition of inorganic phospate (Pi) and hypoxanthine or inosine (the 'inosine cycle'), steady-state levels of the intermediates are observed and the cycle 'turns over' as far as 5-phosphoribosyl-1-pyrophosphate is being consumed. In the presence of ATP, which acts both as an activator of IMP-GMP-specific 5'-nucleotidase and as substrate of nucleoside mono- and di-phosphokinases, no IDP and ITP are formed. The inosine cycle is further favored by the extremely low xanthine oxidase activity. Evidence is presented that ribose 1-phosphate needed to salvage pyrimidine bases in rat brain may arise, at least in part, from the 5-phosphoribosyl-1-pyrophosphate hydrolysis as catalyzed by the inosine cycle, showing that it may function as a link between purine and pyrimidine salvage. When the cycle is started by addition of Pi and guanine (the 'guanosine cycle'), xanthine and xanthosine are formed, in addition to GMP and guanosine, showing that the guanosine cycle 'turns over' in conjunction with the recycling of ribose 1-phosphate for nucleoside interconversion. In the presence of ATP, GDP and GTP are also formed, and the velocity of the cycle is drastically reduced, suggesting that it might metabolically modulate the salvage synthesis of guanyl nucleotides.
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PMID:The purine nucleoside cycle in cell-free extracts of rat brain: evidence for the occurrence of an inosine and a guanosine cycle with distinct metabolic roles. 1278 25