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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)

A procedure is presented for the rapid purification of a 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from potato tubers, involving ammonium sulphate fractionation and chromatography on phosphocellulose, DEAE-cellulose and Sephadex G-75. Application of this procedure results in a 6000-fold purification of the 5'-nucleotidase and the final preparations are virtually homogeneous, yielding only one protein band on electrophorsis in polyacrylamide gels in non-dissociating or dissociating conditions. The 5'-nucleotidase has a molecular weight of 50 000 from gel filtration experiments. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified 5'-nucleotidase reveals one major band of molecular weight 25 000. The 5'-nucleotidase is competitively inhibited by cyclic nucleotides, having micromolar Ki values for cyclic AMP and cyclic GMP at pH 5.0 and pH 8.0. The enzyme has a pH optimum of 5.0 with 5'-GMP as substrate. While 5'-AMP and 3'-AMP are hydrolyzed at comparable rates at pH 5.0, at pH 8.0 the rate of hydrolysis of 3'-AMP is only 4% of that with 5'-AMP. ADP, ATP and 2'-AMP are very poor substrates for the enzyme. The nucleotidase has micromolar Km values for nucleoside 5'-monophosphates other than 5'-NMP. A wide variety of divalent cations activate the 5'-nucleotidase.
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PMID:Purification and characterization of a cyclic nucleotide-regulated 5'-nucleotidase from potatoe. 23 99

Perfused rat hearts catalyze the hydrolysis of AMP added to the perfusion fluid at a rate of 35 mumol/g dry weight/min. The activity is specific for 5'-nucleoside monophosphates, little activity being observed with 2' and 3'-AMP. The enzyme exhibits Michaelis-Menten kinetics in situ and is inhibited competitively by adenosine-5'-alpha, beta-methylene diphosphonate (Ki = 13 muM). This, as well as the nucleotide specificity, confirms that the hydrolysis is catalyzed by 5'-nucleotidase. The maximum activity of 5'-nucleotidase in perfused hearts is equal to or greater than that found in heart homogenates; thus, all of the enzyme is accessible to AMP added externally. Hydrolysis of endogenous AMP was studied in the perfused heart. Under aerobic conditions hearts contain very low amounts of purine nucleosides, and little or no nucleoside is found in the effluent perfusate. Under anaerobic conditions hearts accumulate adenosine, inosine, and hypoxanthine and release all three substances into the perfusate. Hydrolysis of externally added AMP was also observed in perfused skeletal muscle and liver, at rates of 10 and 17 mumol/g dry weight/min, respectively.
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PMID:Studies of 5'-nucleotidase in the perfused rat heart. Including measurements of the enzyme in perfused skeletal muscle and liver. 97 76

Specific binding of [3H]AMP to rat hepatocytes and their plasma membranes was studied. It was shown that the time course of this binding reached a maximum within the first 15 seconds. An equilibrium binding study revealed the presence of a single class of binding sites with Kd of 20 microM both in hepatocytes and in plasma membranes. The [3H]AMP binding sites were inactivated by treatment with trypsin as well as by heating. 5'-Phosphorylated derivatives of adenosine (ATP, ADP) effectively competed with [3H]AMP for the binding sites, while adenosine, beta-glycerophosphate and 3'-AMP were inactive. The binding of [3H]AMP increased by 400% in the presence of concanavalin A, a specific inhibitor of plasma membrane 5'-nucleotidase. It was concluded that the catalytic center of 5'-nucleotidase is a receptor for adenine nucleotides.
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PMID:[Interaction of [3H]AMP with liver cells and their plasma membranes]. 187 49

Hydrolysis of 5'-AMP by 5'-nucleotidase is a possible source of adenosine in the kidney. A renal membrane-bound ecto-5'-nucleotidase has been previously described. The present study deals with the catalytic properties of a 5'-AMP phosphohydrolase partially purified from high-speed supernatants of rat kidney homogenates. It exhibits phosphatase activity toward 5'-AMP, 5'-IMP, and 5'-GMP, but not toward 2'- and 3'-AMP and corresponds therefore to a 5'-nucleotidase. The hydrolysis of 5'-AMP by the soluble 5'-nucleotidase requires divalent cations. Maximal activity is reached with 10 microM of either Mn2+ or Co2+, whereas half-maximal activity is obtained with approximately 400 microM Mg2+. The soluble 5'-nucleotidase exhibits Michaelis-Menten kinetics with a Km of 9.5 microM for 5'-AMP. In the presence of 1 mM of free Mg2+, physiological concentrations of ATP provoke an increase of the Km for 5'-AMP and a decrease of Vmax. An increase of the pH of 0.4 units in the pH range 6.4-7.4 roughly doubles the rate of hydrolysis of 5'-AMP. The effects of ATP and of the pH are compatible with a role of the renal soluble 5'-nucleotidase in the hydrolysis of 5'-AMP and in the production of adenosine during hypoxia.
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PMID:An ATP-inhibited soluble 5'-nucleotidase of rat kidney. 283 Jul 90

1. 5'-Nucleotidase activity was obtained in a soluble form after treatment of a particulate fraction from Ehrlich ascites-tumour cells with deoxycholate. The relative rates of hydrolysis of 6-thioinosine 5'-phosphate, UMP, AMP, CMP, GMP, IMP, xanthosine monophosphate, thymidine monophosphate and 2',3'-AMP were 180, 129, 100, 93, 83, 79, 46, 41 and 3 respectively. 2. Values found for the Michaelis constant were: AMP, 67+/-12mum; IMP, 111+/-8mum; GMP, 93mum. 3. ATP and thymidine triphosphate were competitive inhibitors of AMP hydrolysis (inhibitor constants 0.4 and 4.8mum respectively); UTP, GTP and CTP were mixed competitive and non-competitive inhibitors. Thymidine triphosphate was a competitive inhibitor of IMP hydrolysis (inhibitor constant 14.4mum) and ATP, UTP and GTP showed mixed competitive and non-competitive inhibition. 4. ATP, thymidine triphosphate, UTP, GTP and CTP did not completely inhibit hydrolysis of AMP, IMP and UMP; the concentrations of ATP required to inhibit AMP and IMP hydrolysis by 50% were 12 and 230mum respectively. 5. Non-hyperbolic curves relating activity to UMP concentration were obtained in the presence and absence of triphosphates. 6. After fractionation on Sephadex G-200 columns a single peak of 5'-nucleotidase activity (particle weight 120000-125000) was obtained with AMP, IMP and GMP as substrates. UMP hydrolysis was catalysed by enzyme in this peak and in two slower peaks corresponding to apparent particle weights of 32000 and 16000; a single component (particle weight 120000), reacting with UMP and insensitive to UTP inhibition, was obtained when the column was eluted with buffer containing 1mm-UMP. 7. The possible significance of the results in the regulation of tumour-cell 5'-nucleotidase is discussed.
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PMID:Inhibition of 5'-nucleotidase from Ehrlich ascites-tumour cells by nucleoside triphosphates. 577 89

A previously unknown 5'nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) (5'-Nase) specific for orotidine 5'-monophosphate (OMP) hs been discovered. This enzyme orotidine 5'-monophosphate phosphohydrolase (OMPase), was isolated from mouse liver microsomes as a separate entity from the nonspecific 5'-Nase. OMPase was partially purified and is shown to cleave OMP to orotidine and inorganic phosphate. The enzyme has negligible activity towards UMP, CMP, dTMP, AMP, IMP, GMP, XMP, 6-azauridine 5'-monophosphate, 1-beta-D-ribofuranosylbarbituric acid 5'-monophosphate (BMF), 2'-UMP, 3'-UMP, 2'-AMP, 3'-AMP, ribose 5-phosphate and beta-glycerophosphate, all of which--with the exception of the 2' or 3' monophosphates, ribose 5'-phosphate, and beta-glycerophosphate--are substrates for 5'-Nase. Both enzymes are inhibited by NaF, but only OMPase is inhibited by SF reagents. OMPase is not inhibited by orotidine, orotate, BMP, concanavalin A, or tetramisole (an alkaline phosphatase inhibitor). OMPase had a Mr 53,000, Km value of 1 mM for OMP, and Vmax value of 49 nmol/min . mg of protein at the present stage of purification. OMPase activity has also been detected in various mammalian tissues including normal human tissues, human tumor xenografts, lymphocytes, and rat liver. OMPase may be responsible, in part, for the low levels of intracellular "free" OMP and for orotidine accumulation in cells treated with 6-azauridine and patients suffering from aortic aciduria.
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PMID:Isolation and partial characterization of a 5'-nucleotidase specific for orotidine-5'-monophosphate. 628 Jan 63

Examination of prostasomes, isolated from human seminal plasma, showed that there was very little remaining paranitrophenylphosphatase activity when assayed in the presence of 10 mmol/l of tartrate and 2 mmol/l of levamisole. Under these conditions it was possible to study the prostasome membrane-bound 5'-nucleotidase activity, which was unaffected by these two inhibitors. The activity was considered to be located at the external surface of the prostasome membrane and a 50-60% increase in activity was obtained by the addition of 0.05% Triton X-100. The prostasome membrane-linked 5'-nucleotidase readily hydrolysed 5'-AMP. Two other 5'-nucleoside monophosphates, 5'-IMP and 5'-GMP, were also hydrolysed, but more slowly; 2'- or 3'-AMP were practically not attacked. The prostasome membrane-linked 5'-nucleotidase obeyed Michaelis-Menten kinetics. Apparent Km for 5'-AMP was 11.2 +/- 2.1 mumol/l and Vmax 64.7 +/- 11.4 nmol/mg protein/min. These figures were somewhat changed in presence of 0.05% Triton X-100, the Km value being reduced by 30% and the Vmax value increased by 60%. Adenosine 5' (alpha, beta methylene) diphosphate (100 mumol/l), Ni2+ (10 mmol/l) and concanavalin A (20 micrograms/ml) were all potent inhibitors of the prostasome membrane-linked 5'-nucleotidase.
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PMID:Characteristics of membrane-bound 5'-nucleotidase on human prostasomes. 822 68

We previously demonstrated that extracellular adenine nucleotides induced cyclic AMP elevation through local adenosine production at the membrane surface and subsequent activation of adenosine A(2A) receptors in NG108-15 cells. Furthermore, the adenosine formation was found to be mediated by an ecto-enzyme distinct from the ecto-5'-nucleotidase (CD73). In this study, we investigated the properties of the ecto-AMP phosphohydrolase activity in NG108-15 cells. NG108-15 cells hydrolyzed AMP to adenosine with the K:(M:) value of 18.8+/-2.2 microM and V(max) of 5.3+/-1.6 nmol min(-1) 10(6) cells(-1). This activity was suppressed at pH 6.5, but markedly increased at pH 8.5. The AMP hydrolysis was blocked by levamisole, an alkaline phosphatase (ALP) inhibitor. NG108-15 cells released orthophosphate from 2'- and 3'-AMP as well as from ribose-5-phosphate and ss-glycerophosphate, indicating that NG108-15 cells express ecto-ALP. The cyclic AMP accumulation induced by several adenine nucleotides was inhibited by levamisole, p-nitrophenylphosphate and ss-glycerophosphate, with a parallel decrease in the extracellular adenosine formation. Reverse transcriptase polymerase chain reaction analysis revealed that NG108-15 cells express mRNA for the tissue-nonspecific isozyme of ALP. These results demonstrate that AMP phosphohydrolase activity in NG108-15 cells is due to ecto-ALP, and suggest that this enzyme plays an essential role for the P1 antagonist-sensitive ATP-induced cyclic AMP accumulation in NG108-15 cells.
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PMID:Ecto-alkaline phosphatase in NG108-15 cells : a key enzyme mediating P1 antagonist-sensitive ATP response. 1113 45

Two forms (F-I and F-II) of 5'-nucleotidases (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N(6)-(Delta(2)-isopentenyl)adenosine 5'-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5'-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K(m) values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5'-phosphates, AMP being the preferred substrate. N(6)-(Delta(2)-isopentenyl)Adenosine 5'-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3'-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5'-nucleotidases may play an important role in regulating levels of "active cytokinin" in plant cells.
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PMID:Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL. 1666 1