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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)

Acetylcholine and ATP are costored and coreleased during synaptic activity at the electric organ of Torpedo. It has been suggested that released ATP is converted to adenosine at the synaptic cleft, and in turn this nucleoside would depress the evoked release of acetylcholine. In the present communication we have used a chemiluminescent reaction that let us to monitor continuously the presence of adenosine in this preparation. The chemiluminescent reaction is based on the conversion of adenosine into uric acid and H2O2 by adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase enzymes. The hydrogen peroxide has been detected by peroxidase-luminol mixture. The reaction has a sensitivity on the picomol range and discerned between Adenosine, AMP, ADP, and ATP. We have developed this technique in the hope of understanding whether adenosine is released during synaptic activity or it comes from the released ATP. We have studied the release or formation of adenosine in fragments of the electric organ and in isolated cholinergic nerve terminals obtained from it. In both conditions we have followed the effect of potassium stimulation upon the detection of adenosine. Potassium stimulation increased the extracellular adenosine either in slices or the synaptosomal fraction of Torpedo electric organ. The presence of alpha, beta-methylene ADP, an inhibitor of 5'-nucleotidase, inhibits the detection of adenosine, suggesting that extracellular adenosine is a consequence of ectocellular dephosphorylation of released ATP.
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PMID:The release of adenosine at the electric organ of Torpedo. A study using a continuous chemiluminescent method. 232 27

The functional state of the gastric mucosa in dogs after 12.7 Gy abdominal X-ray irradiation was examined. During the first 7 days after irradiation, hypochylia, decrease of proteolytic activity and the H+ (hydrogen-ion) production in the gastric juice was observed. The gastric mucosal cells of rats were examined after the irradiation with 12 Gy to find out the reason for hypochylia. Possibly the reduction of the number of the gastric mucosal cells and their protein content one day after the irradiation, as well as functional disorders of the outer membrane--expressed by the decrease of the intracellular K+ (potassium-ion) level and the ATPase and 5'-nucleotidase activity--may be the reasons for the hypochylia after irradiation.
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PMID:[Mechanisms of the inhibition of the functional activity of the stomach in acute radiation sickness]. 274 6

A spectrophotometric method is described for the determination of 5'-nucleotidase. In combination with the enzymes nucleoside phosphorylase and xanthine oxidase, inosine, formed by hydrolysis of 5'-IMP by 5'-nucleotidase, is cleaved phosphorolytically to hypoxanthine, which is oxidized to uric acid. In the presence of ethanol, the hydrogen peroxide formed is reduced by catalase and equivalent amounts of acetaldehyde are produced. The aldehyde is dehydrogenated (NADP-dependent) by aldehyde dehydrogenase and the production rate of NADPH is recorded at 334 nm. The inhibition of the unspecific cleavage of 5'-IMP by phosphatases is examined critically.
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PMID:A new spectrophotometric method for the determination of 5'-nucleotidase. 625 57

We describe a one-step kinetic method for the determination of 5'-nucleotidase (EC 3.1.3.5). Inosine is formed by the hydrolysis of inosine 5'-monophosphate which is catalyzed by seric 5'-nucleotidase, and then is converted to hypoxanthine by nucleoside phosphorylase. Two moles of hydrogen peroxide are formed for each mole of hypoxanthine oxidized to urate by xanthine oxidase. The rate formation of hydrogen peroxide is monitored at 510 nm using the oxidation of the chromogenic system 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone in the presence of peroxidase. beta-Glycerophosphate inhibits the unspecific cleavage of the substrate by alkaline phosphatases. Inorganic phosphate is added to improve the reagent stability, and ferrocyanide to reduce bilirubin interference. Automation of the technique requiring 20 microliter of serum on a centrifugal analyzer is also described.
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PMID:A one-step determination of serum 5'-nucleotidase using a centrifugal analyzer. 627 35

The continuous spectrophotometric assay of 5'-nucleotidase originally described by Heinz et al. ((1980) J. Clin. Chem. Clin. Biochem. 18, 781-788) was modified and fully automated on a Kem-O-Mat transfer analyzer, using inosine 5'-monophosphate as substrate. The reaction product was hydrogen peroxide and the reduction of NADP was observed for 10 minutes at 340 nm and at a reaction temperature of 30 degrees C. The different factors involved in the enzyme reaction were checked, including the substrate concentration, reaction rate, linearity and substrate preservation. Normal values ranged from 1 to 13 U/l. Between-day reproducibility was estimated with two different commercial control sera, and the coefficient of variation was 5% for the upper limit of normal activity (23 U/l). There was good agreement between the present method and a semi-automatic colorimetric technique (for 100 sera tested by both methods, the correlation coefficient was 0.974 and the regression line equation, y = 0.85 x- 1.5). Despite the lengthy reagent mixture preparation procedure, the method permitted assay of 50 samples per hour. The occurrence of high serum blanks in certain pathological states is discussed.
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PMID:Continuous assay of serum 5-nucleotidase activity with inosine 5'-monophosphate as substrate and total automation using a transfer-analyzer (Kem-O-Mat). 631 43

5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) occurs in bull seminal plasma in multiple forms. The heterogeneity does not reflect the existence of true isoenzymes, but is due to the association of the enzyme with particulate material and to molecular aggregation phenomena. Addition of detergents to native bull seminal plasma prevents molecular aggregation, solubilizes the particulate form of the enzyme, and results in the appearance of a single molecular form of the enzyme. Enzyme purification can be achieved after three chromatographic steps which involve negative adsorption of 5'-nucleotidase activity on DEAE-Sephadex A-50 followed by two affinity chromatographies on concanavalin A-Sepharose 4B and ADP-agarose. The enzyme appears to be a dimeric glycoprotein. Some properties of the enzyme, including substrate specificity and the effects of hydrogen ion concentration and of various divalent cations, are reported.
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PMID:5'-nucleotidase from bull seminal plasma. 631 64

Freeze-substituted rat liver embedded in glycol methacrylate (GMA) has been used to demonstrate the activities of several enzymes. The following enzymes could be detected in GMA-sections by the indicated histochemical procedure(s): 5'-nucleotidase (lead salt, cerium-diaminobenzidine), alkaline phosphatase (indoxyl-tetrazolium salt), catalase (diaminobenzidine), acid phosphatase (diazonium salt), lactate dehydrogenase (tetrazolium salt) and glutamate dehydrogenase (tetrazolium salt). The activities of all these enzymes were dramatically decreased compared with the activities demonstrated in unfixed cryostat sections, with the exception of catalase. The activities of the following enzymes could not be detected in GMA-sections: glucose-6-phosphate dehydrogenase (tetrazolium salt), xanthine oxidoreductase (tetrazolium salt), D-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide) and glucose-6-phosphatase (cerium-diaminobenzidine). The possible role of restricted penetration of reagents into the resin was studied by measuring cytophotometrically the enzyme activities in GMA-sections of 3 and 6 microns in thickness. For all the enzymes that could be detected, the 6 microns:3 microns ratio varied from 1.4 to 2.7. An eventual retarded penetration of reagents into the resin was investigated by measuring cytophotometrically the amount of final reaction product during incubation for acid phosphatase and glutamate dehydrogenase activities. In both cases linear relationships without a lag phase were found for the specific enzyme activities with incubation time. Chemical denaturation of proteins or masking of active sites in proteins due to embedding in the resin monomer may be considered to be the main cause of decreased enzyme activities.
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PMID:Quantitative aspects of enzyme histochemistry on sections of freeze-substituted glycol methacrylate-embedded rat liver. 827 44

The effect of storage of unfixed cryostat sections from rat liver for 4 h, 24 h, 3 days and 7 days at -25 degrees C was studied on the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidoreductase, glutamate dehydrogenase, succinate dehydrogenase (all demonstrated with tetrazolium salt procedures), glucose-6-phosphatase (cerium-diaminobenzidine method), 5'-nucleotidase (lead salt method), dipeptidyl peptidase II, acid phosphatase (both simultaneous azo coupling methods), D-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure) and catalase (diaminobenzidine method). The effect of drying of the cryostat sections at room temperature for 5 and 60 min was investigated as well. The enzyme activities were quantified by cytophotometric measurements of test and control reactions. The test minus control reaction was taken as a measure for specific enzyme activity. It was found that the activities of all the enzymes investigated, with one exception, were affected neither by storage of the cryostat sections at -25 degrees C for up to 7 days, nor by drying of the sections at room temperature for up to 60 min. The exception was xanthine oxidoreductase, whose activity was reduced by 20% after 5 min drying of sections or after 4 h storage. Therefore, only incubations for xanthine oxidoreductase activity have to be performed immediately after cutting cryostat sections, whereas for the other enzymes a considerable margin appears to exist.
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PMID:The effects of storage on the retention of enzyme activity in cryostat sections. A quantitative histochemical study on rat liver. 846 85

Various 5'-nucleotidases (EC 3.1.3.5) exist in vertebrate tissues. The sequence and cDNA cloning of the membrane-bound ecto-5'-nucleotidase (e-N) and one of the cytosolic isoenzymes, IMP-preferring (c-N-II), but not the cytosolic AMP-preferring form (c-N-I), have been reported. While c-N-II has a broad tissue distribution, c-N-I is found only in vertebrate heart. The published data on substrate specificity involve mainly the naturally occurring nucleoside monophosphates, without a systematic structure-activity relationship study. In the present study we have used a series of AMP and IMP analogues to examine the structure-activity relationship for c-N-I and c-N-II in detail. The rank order of activity of the test compounds differed substantially between c-N-I and c-N-II. c-N-I and c-N-II varied with respect to the following interactions with substrate: (1) hydrogen-bond formation with the substituent in the 6-position of the purine ring (a donor-type with c-N-I and an acceptor-type with c-N-II); and (2) hydrophobic attraction of the 6-position unsubstituted purine ring (more pronounced with c-N-I than with c-N-II). No better substrate than 5'-AMP was found for c-N-I. We propose that c-N-I functions as an AMP-binding protein in the myocardial cell with an important role during ischaemic ATP breakdown when AMP accumulates rapidly.
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PMID:Structure-activity relationship of cytoplasmic 5'-nucleotidase substrate sites. 861 51

AMP analogues modified at various positions of the molecule were checked as substrates for the two soluble isoforms of 5'-nucleotidase from human seminal plasma. These isoforms were isolated to near homogeneity by affinity chromatographies. AMP derivatives were differently dephosphorylated by both the isoforms depending on the site of modification in the natural compound. Changes in the phosphate moiety reduced significantly hydrolysis by the IMP-preferring form, whereas the AMP-preferring form was less affected. The AMP-preferring form was characterized by a relatively broad specificity toward substrate analogues indicating that the binding domains for the phosphate moiety of these isoforms are not identical. Substitutions at the C-8 adenine base reduced the hydrolysis rate of both the enzymes and variations of the syn-anti conformational equilibrium resulted in different effects on catalysis by both forms. Therefore, the orientation of the heterocyclic base around the glycosidic bond may not be the crucial factor affecting binding and catalytic activity. Hydrogen bonding potential of base N-7 was essential for the binding and catalysis of the IMP- but not of the AMP-preferring form. This was the most striking difference between the studied isoforms. Modifications and substitutions of 6-amino function, better accepted by the IMP-preferring form than by the AMP-preferring form, indicated that no essential hydrogen bonding is required for catalytic activity. The binding was however significantly slowed in 6-SH-PuMP. Hydrogen bonding potential of N-1 was significant for the hydrolysis rate of the IMP- but not of the AMP-preferring form. We suggest that these human seminal plasma isoforms of soluble 5'-nucleotidase, characterized by unique features, may represent the tissue-specific expression of the polymorphic gene.
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PMID:Activity of IMP- and AMP-preferring isoforms of 5'-nucleotidase from human seminal plasma with AMP analogues. 997 47


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