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

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on Escherichia coli some 30 to 60% of the initial E. coli RNA-ribose disappeared as cell-associated orcinol-positive material. The levels of RNA-ribose in the suspending buffer after growth together with the RNA-ribose used for bdellovibrio DNA synthesis accounted for 50% or less of the missing RNA-ribose. With intraperiplasmic growth in the presence of added U-14C-labeled CMP, GMP, or UMP, radioactivity was found both in the respired CO2 and incorporated into the bdellovibrio cell components. The addition of exogenous unlabeled ribonucleotides markedly reduced the amounts of both the 14CO2 and 14C incorporated into the progeny bdellovibrios. During intraperiplasmic growth of B. bacteriovorus on [U-14C]ribose-labeled E. coli BJ565, ca. 74% and ca. 19% of the initial 14C was incorporated into the progeny bdellovibrios and respired CO2, respectively. Under similar growth conditions, the addition of glutamate substantially reduced only the 14CO2; however, added ribonucleotides reduced both the 14CO2 and the 14C incorporated into the progeny bdellovibrios. No similar effects were found with added ribose-5-phosphate. The distribution of 14C in the major cell components was similar in progeny bdellovibrios whether obtained from growth on [U-14C]ribose-labeled E. coli BJ565 or from E. coli plus added U-14C-labeled ribonucleotides. After intraperiplasmic growth of B. bacteriovorus on [5,6-3H-]uracil-[U-14C]ribose-labeled E. coli BJ565 (normal or heat treated), the whole-cell 14C/3H ratio of the progeny bdellovibrios was some 50% greater and reflected the higher 14C/3H ratios found in the cell fractions. B. bacteriovorus and E. coli cell extracts both contained 5'-nucleotidase, uridine phosphorylase, purine phosphorylase, deoxyribose-5-phosphate aldolase, transketolase, thymidine phosphorylase, phosphodeoxyribomutase, and transaldolase enzyme activities. The latter three enzyme activities were either absent or very low in cell extracts prepared from heat-treated E. coli cells. It is concluded that during intraperiplasmic growth B. bacteriovorus degrades some 20 to 40% of the ribonucleotides derived from the initial E. coli RNA into the base and ribose-1-phosphate moieties. The ribose-1-phosphate is further metabolized by B. bacteriovorus both for energy production and for biosynthesis, of non-nucleic acid cell material. In addition, the data indicate that during intraperiplasmic growth B. bacteriovorus can metabolize ribose only if this compound is available to it as the ribonucleoside monophosphate.
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PMID:Metabolism of RNA-ribose by Bdellovibrio bacteriovorus during intraperiplasmic growth on Escherichia coli. 36 99

Two enzymatic activities that split diadenosine triphosphate have been reported in Escherichia coli: a specific Mg-dependent bis(5'-adenosyl) triphosphatase (EC 3.6.1.29) and the bis(5'-adenosyl) tetraphosphatase (EC 3.6.1.41). In addition to the activities of these two enzymes, a different enzyme activity that hydrolyzes dinucleoside polyphosphates is described. After purification and study of its molecular and kinetic properties, we concluded that it corresponded to the 5'-nucleotidase (EC 3.1.3.5) that has been described in E. coli. The enzyme was purified from sonic extracts and osmotic shock fluid. From sonic extracts, two isoforms were isolated by chromatography on ion-exchange Mono Q columns; they had a molecular mass of about 100 kilodaltons (kDa). From the osmotic shock fluid, a unique form of 52 kDa was recovered. Mild heating transformed the 100-kDa isoform to a 52-kDa form, with an increase in activity of about threefold. The existence of a 5'-nucleotidase inhibitor described previously, which associates with the enzyme and is not liberated in the osmotic shock fluid, may have been responsible for these results. The kinetic properties and substrate specificities of both forms (52 and 100 kDa) were almost identical. The enzyme, which is known to hydrolyze AMP and uridine-(5')-diphospho-(1)-alpha-D-glucose, but not adenosine-(5')-diphospho-(1)-alpha-D-glucose, was also able to split adenosine-(5')-diphospho-(5)-beta-D-ribose, ribose-5-phosphate, and dinucleoside polyphosphates [diadenosine 5',5'''-P1,P2-diphosphate,diadenosine 5',5'''-P1,P3-triphosphate, diadenosine 5',5'''-P1,P4-tetraphosphate, and bis(5'-guanosyl) triphosphate]. The effects of divalent cations and pH on the rate of the reaction with different substrates were studied.
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PMID:Hydrolysis of bis(5'-nucleosidyl) polyphosphates by Escherichia coli 5'-nucleotidase. 255 71

Susceptibilities to snake venom 5'-nucleotidase (EC 3.1.3.5) have been evaluated for several 8-substituted analogues of 5'-GMP with varying populations of syn/anti conformations about the glycosidic bond. Improved syntheses of some of these are described, including direct chlorination of 5'-GMP to give 8-chloro-5'-GMP, a procedure which should be applicable to other purine nucleotides. The conformations of the various analogues were determined by means of 1H NMR spectroscopy, with particular emphasis on the glycosidic bond conformations. All the 8-substituted derivatives of 5'-GMP were relatively poor substrates of 5'-nucleotidase. This was shown to result largely from steric effects and the nature of the 8-substituent, and consistent with a requirement for the anti conformation. Although ribose-5-phosphate was not a substrate, it was a weak inhibitor, and its inhibitory properties account in part for the weak inhibitory properties of the 8-substituted 5'-GMP, and other, analogues. Attention is drawn to the hitherto largely neglected differences in properties of 5'-nucleotidases from different sources and their relevance to the present findings.
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PMID:Conformation about the glycosidic bond and susceptibility to 5'-nucleotidase of 8-substituted analogues of 5'-GMP. 632 8

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

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

5'-nucleotidases (EC 3.1.3.5) catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides as well as complex nucleotides, such as uridine 5'-diphosphoglucose (UDP-glucose), nicotinamide adenine dinucleotide and flavin adenine dinucleotide, to their corresponding nucleosides plus phosphate. These enzymes have been found in diverse species in intracellular and membrane-bound, surface-localized forms. Soluble forms of 5'-nucleotidases belong to the ubiquitous haloacid dehalogenase superfamily (HADSF) and have been shown to be involved in the regulation of nucleotide, nucleoside and nicotinamide adenine dinucleotide (NAD+) pools. Despite the important role of 5'-nucleotidases in cellular metabolism, only a few of these enzymes have been characterized in the Gram-positive bacterium Bacillus subtilis, the workhorse industrial microorganism included in the Food and Drug Administration's GRAS (generally regarded as safe) list. In the present study, we report the identification of a novel 5'-nucleotidase gene from B. subtilis, yutF, which comprises 771 bp encoding a 256-amino-acid protein belonging to the IIA subfamily of the HADSF. The gene product is responsible for the major p-nitrophenyl phosphatase activity in B. subtilis. The yutF gene was overexpressed in Escherichia coli, and its product fused to a polyhistidine tag was purified and biochemically characterized as a soluble 5'-nucleotidase with broad substrate specificity. The recombinant YutF protein was found to hydrolyze various purine and pyrimidine 5'-nucleotides, showing preference for 5'-nucleoside monophosphates and, specifically, 5'-XMP. Recombinant YutF also exhibited phosphohydrolase activity toward nucleotide precursors, ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate. Determination of the kinetic parameters of the enzyme revealed a low substrate specificity (Km values in the mM concentration range) and modest catalytic efficiencies with respect to substrates. An initial study of the regulation of yutF expression showed that the yutF gene is a component of the yutDEF transcription unit and that YutF overproduction positively influences yutDEF expression.
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PMID:Identification, Heterologous Expression, and Functional Characterization of Bacillus subtilis YutF, a HAD Superfamily 5'-Nucleotidase with Broad Substrate Specificity. 2790 99

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