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)

5'-Nucleotidase has been purified from rat glioblastoma cells (Rugli cells). The enzyme has been solubilized from plasma membranes by using Triton X-100 and CHAPS. Two affinity chromatographies on concanavalin A and 5'-AMP-Sepharose render the purified enzyme with a high specific activity (76.36 mumol AMP.min-1.mg-1). The purified enzyme gives a single polypeptide band on SDS-PAGE with an apparent molecular mass of 74 kDa. Active forms with an apparent molecular mass of 135 kDa and 268 kDa are observed when the purified enzyme is analyzed by gel filtration in the presence of either 0.6% sodium deoxycholate or 0.1% Triton X-100, respectively. The purified 5'-nucleotidase presents optimum activity at pH 7.8-8.1 either in the presence or in the absence of Mg2+. A linear Arrhenius plot is observed in the 25-46 degrees C temperature range and an activation energy of 33.7 KJ/mol is calculated. The enzyme is inhibited by EDTA; the activity is partially restored by different divalent cations as Zn2+, Mn2+, and Co2+. The hydrolysis of nucleosides 5'-monophosphate shows Michaelis kinetic. The enzyme is inhibited by nucleosides di- and triphosphate. 5'-Nucleotidase is a glycoprotein, being its activity inhibited at different extent by various lectins.
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PMID:Isolation and characterization of the ecto-5'-nucleotidase from a rat glioblastoma cell line. 148 Jan 62

The controversial subject of mitochondrial 5'-nucleotidase in the liver was studied employing density gradient fractionation combined with a method for analyzing the distribution profiles of marker enzymes based on multiple regression analysis. Triton WR-1339 was used to improve the separation of mitochondria from lysosomes by the gradient centrifugation technique. Adenosine production was examined further using acetate to increase intramitochondrial AMP, and thus adenosine production, in incubations with gradient centrifugation-purified mitochondria. Distribution analysis of the crude homogenate showed that 5'-nucleotidase activity exists in the mitochondrial fraction. To increase the resolution of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied. In this case the relative mitochondrial activity decreased but 5'-nucleotidase activity was still clearly detectable. The mitochondrial 5'-nucleotidase exhibited a Km of 94 microM and a Vmax of 31 nmol/min per mg protein for AMP. The kinetic data for the Mg2+, ATP, ADP and AOPCP sensitivity of the enzyme showed that it differs from the plasma membrane, lysosome and cytosol 5'-nucleotidases. AOPCP was only a moderate inhibitor, and ATP was a more potent inhibitor than ADP at a 1 mM concentration. The enzyme also showed a requirement of Mg2+. Acetate caused the conversion of intramitochondrial adenylates to AMP and the formation of adenosine. Adenosine concentration increased in the extramitochondrial space in a time-dependent manner, but only trace amounts of nucleotides were detected. The data show that 5'-nucleotidase activity producing adenosine exists in rat liver mitochondria and a concentration-dependent adenosine output from mitochondria by diffusion or facilitated diffusion is also suggested.
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PMID:5'-Nucleotidase activity and adenosine production in rat liver mitochondria. 155 Aug 32

A preliminary examination for the purification and characterization of 5'-nucleotidase of fish muscle was carried out and the following results were obtained. 1. The activities of 5'-nucleotidase in the muscles of marine vertebrates and invertebrates (total 11 species) were determined. The highest activity of 5'-nucleotidase was found in Blackrock fish Sebastes inermis, which was then used as a material for estimation of subcellular distribution and solubilization of the enzyme. 2. The 5'-nucleotidase of ordinary muscle of the fish Sebastes inermis was found in nuclear, microsomal and cytosolic fractions. About half of the total activity was found in the nuclear fraction, whereas the highest specific activity was observed in the microsomal fraction. 3. Complete solubilization of the enzyme was attained by using a high concentration of detergent such as Triton X-100, CHAPS, octylglucoside, octylthioglucoside and sodium deoxycholate, suggesting that the enzyme was tightly bound to the membrane. 4. Based on the results of solubility and stability tests, Triton X-100 seemed suitable for solubilizing 5'-nucleotidase from the membrane. 5. Microsomal 5'-nucleotidase was an Mg(2+)-activated enzyme, and no inactivation was observed up to 50 mM of Mg2+.
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PMID:Distribution of 5'-nucleotidase in muscle of some marine fishes. 161 35

In the lymphocytes infected in vitro with BLV (bovine leukemia virus) the contents of Ca2+ and Mg2+ were determined using roentgen microanalyser JXA-5 A Joel-form (Japan). In the smears prepared from these cells the activity of enzyme markers of cell membranes i.e. alkaline phosphatase (AP - EC 3.1.3.1), 5'-nucleotidase (5'-NT - EC 3.1.3.5) and adenosine-triphosphatases - Ca2+ and Mg2+ dependent (ATP-ase - EC 3.6.1.3) was determined. The decrease in AP and ATP-assess activity and increase in 5'-NT in the membranes of leukemic lymphocytes were observed. During these changes the increase in Ca2+ and decrease in Mg2+ ions occurred. These processes lead to clear disturbances in the metabolism of cells transformed by the neoplasm. The effect of this phenomenon is probably the opening of calcium canals with the following cytoplasmatic hypercalcemia. It's very destructive for the change in permeability of the membrane of lymphocytes.
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PMID:[The content of Ca2+ and Mg2+ ions and membrane enzyme activity (AP, 5'-NT, ATPases) in the lymphocytes infected in vitro with bovine leukemia virus]. 166 9

Soluble low Km 5'-nucleotidase from human seminal plasma has been purified to homogeneity by one affinity and two gel-filtration chromatographic steps. The pure enzyme had a specific activity of 2000 nmol min-1 mg-1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified low Km 5'-nucleotidase revealed a single polypeptide band of 40 +/- 7 kDa and a tetrameric structure of 160 +/- 10 kDa has been proposed for the native enzyme. The kinetic properties of low Km 5'-nucleotidase have been determined and rather unique characteristics have been found for this soluble low Km 5'-nucleotidase: the substrate efficiency was slightly higher for IMP with an optimum pH at 7.5; the enzyme showed an absolute dependence on Mg2+ ions. Ca2+ could replace Mg2+ ions for activity while other divalent cations could not substitute for Mg2+; the enzymes were equally activated by ATP and ADP up to 0.1 mM concentrations. At higher concentrations up to 1 mM, ADP was still an activator while ATP caused a gradual decrease of activation to the native activity. This effect could not be related to the Mg-ATP = complexes since the enzymic preparation Mg(2+)-free still showed the same biphasic pattern of activation.
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PMID:Purification and partial characterization of the soluble low Km 5'-nucleotidase from human seminal plasma. 195 33

5'-Nucleotidase I (N-I) from rabbit heart was purified to homogeneity. After ammonium sulfate precipitation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose, AMP-agarose, and ADP-agarose. The pure enzyme has a specific activity of 318 mumol (mg of protein)-1 min-1. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a subunit molecular weight of 40,000. N-I is activated by ADP but not by ATP, in contrast to the 5'-nucleotidase (N-II) purified by Itoh et al. (1986), which is activated by ATP and, less well, by ADP. N-I displays sigmoidal saturation kinetics in the absence of ADP and hyperbolic kinetics in the presence of ADP. Partially purified N-I was previously shown to prefer AMP over IMP as substrate (Truong et al., 1988); this has been confirmed for pure N-I. Comparison of AMP and ADP concentrations reported to occur in heart with the kinetic behavior of N-I implicates N-I as the enzyme responsible for producing adenosine under conditions leading to a rise in ADP and AMP, such as hypoxia or increased workload. N-I is not activated by the ADP analogue adenosine 5'-methylenediphosphonate (AOPCP) and is only weakly inhibited by relatively high concentrations of AOPCP, in contrast to 5'-nucleotidase from plasma membrane, which is powerfully inhibited by this analogue. N-I shows an absolute dependence on Mg2+ ions. Mn2+ and Co2+ ions can replace Mg2+ ions as activator; Ni2+ and Fe2+ are much less effective, while Ca2+, Ba2+, Zn2+, and Cu2+ fail to activate the enzyme.
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PMID:5'-Nucleotidase I from rabbit heart. 199 69

The plasma membrane fraction of chicken osteoclasts was purified utilizing 20% continuous Percoll gradients. Biochemical marker enzyme analysis (ouabain-sensitive Na+,K(+)-ATPase and 5'-nucleotidase) indicated that plasma membrane enrichment was 11.87-fold and 7.25-fold, respectively, and contamination with mitochondria, endoplasmic reticulum, and lysosomes was low as determined by succinic dehydrogenase, NADH dehydrogenase, and N-acetylglucosaminidase activities, respectively. SDS latency of Na+,K(+)-ATPase and 5'-nucleotidase activities of the isolated plasma membranes revealed that 43-50% of vesicles were sealed, with 10-16% in the inside-out orientation, depending on the membrane fraction used. Electron microscopy confirmed the vesicular nature of the plasma membrane fraction. The plasma membrane Ca2(+)-ATPase had a high-affinity (KCa = 0.22 microM; Vmax = 0.16 mumol/mg per min) and a low-affinity (KCa = 148 microM; Vmax = 0.37 mumol/mg per min) component. Calmodulin (0.12 microM) had no effect on Ca2(+)-ATPase activity. However, trifluoperazine (0.1 mM), a calmodulin antagonist, strongly inhibited especially the high-affinity component of the enzyme. Vanadate and lanthanum also caused inhibition. In the presence of CDTA, a potent Ca2+ and Mg2+ chelating agent, high-affinity Ca2(+)-ATPase activity was abolished, indicating that trace Mg2+ was essential for activity. The Ca2(+)-ATPase substrate curve using ATP showed a high-affinity (Km = 12.3 microM; Vmax = 0.022 mumol/mg per min) and a low-affinity (Km = 43.8 microM; Vmax = 0.278 mumol/mg per min) component. These results demonstrate that osteoclasts have a plasma membrane Ca2(+)-ATPase with characteristics similar to the enzyme responsible for active calcium extrusion in other cells.
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PMID:Characterization of a Ca2(+)-ATPase in osteoclast plasma membrane. 214 47

A (H+ + K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation. The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na+ + K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H+ + K+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (p-NPPase). The (H+ + K+)-ATPase had a pH optimum of 7.4 and was stimulated by Tl+, K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM, respectively, at this pH. On the other hand, monovalent cations such as Na+, Li+ and (CH3)4N+ as well as divalent cations such as Cu2+, Ca2+, Ba2+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently. Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H+ + K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed a major band (greater than or equal to 90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [gamma-32P]-ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H+ + K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.
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PMID:Purification and characterization of (H+ + K+)-ATPase from hog gastric mucosa. 215 97

A soluble 5'-nucleotidase was purified 200-fold from pigeon heart. The enzyme (1) had an apparent molecular mass close to 150 kDa, (2) had a neutral pH optimum and hydrolysed a wide range of nucleoside 5'-monophosphates with a 15-fold preference for AMP over IMP, (3) at near-physiological concentrations of AMP was activated by ADP but not by ATP, (4) was inhibited by high Mg2+ concentration and high ionic strength, (5) was weakly inhibited by p-nitrophenol phosphate and Pi, and (6) was non-competitively inhibited more potently by 5'-deoxy-5'-isobutylthioinosine than by 5'-deoxy-5'-isobutylthioadenosine, but not by [alpha,beta-methylene]ADP. The data show that the enzyme is distinct from the ecto-5'-nucleotidase and from the previously purified IMP-specific 5'-nucleotidase. They also predict that the enzyme is activated during ATP catabolism and hence will generate a more-than-linear increase in the adenosine-formation rate in response to an increase in cytosolic AMP concentration.
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PMID:Partial purification and properties of an AMP-specific soluble 5'-nucleotidase from pigeon heart. 234 53

The effect of lipid peroxidation on the Mg2(+)-independent and Mg2(+)-dependent activity of brain cell membrane 5'-nucleotidase was determined and the affinity of the active sites of Mg2(+)-dependent enzyme for 5'-AMP (substrate) and Mg2+ (activator) was examined. Brain cell membranes were peroxidized at 37 degrees C in the presence of 100 microM ascorbate and 25 microM FeCl2 (resultant) for 10 min. The activity of 5'-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20 +/- 0.10 to 17.5 +/- 1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg2(+)-independent 5'-nucleotidase increased from 0.201 +/- 0.020 in controls to 0.305 +/- 0.028 mumol Pi/mg protein/hr in peroxidized membranes. In the presence of 10 mM Mg2+, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control. In peroxidized preparation, the affinity of active site of Mg2(+)-dependent 5'-nucleotidase for 5'-AMP tripled, as indicated by a significant decrease in Km (Km = 95 +/- 2 microM AMP for control; Km = 32 +/- 2 microM AMP for peroxidized). Vmax was significantly reduced from 3.35 +/- 0.16 in control to 1.70 +/- 0.9 mumoles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg2+ significantly increased (Km = 6.17 +/- 0.37 mM Mg2+ for control; Km = 4.0 +/- 0.31 peroxidized).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lipid peroxidation as the mechanism of modification of brain 5'-nucleotidase activity in vitro. 236 28


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