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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)

We report a rapid and reproducible assay for activity of human erythrocyte pyrimidine 5'-nucleotidase and deoxypyrimidine 5'-nucleotidase. The nucleotides CMP, UMP, dUMP, dCMP or dTMP are individually incubated 30 min at 37 degrees C with erythrocyte hemolysate and 4 mM magnesium chloride in Tris, pH 7.5. Data are provided for standardization of the reaction with each substrate. Individual nucleoside products are assayed in less than 10 min by reversed-phase high-performance liquid chromatography at 280 nm with 0-14% methanol in 0.01 M potassium dihydrogen phosphate. This is the first report of a high-performance liquid chromatographic assay system which allows quantitation of the activity of pyrimidine 5'-nucleotidase isozymes using five individual pyrimidine and deoxypyrimidine nucleotides as the substrates.
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PMID:Assay of human erythrocyte pyrimidine and deoxypyrimidine 5'-nucleotidase by isocratic reversed-phase high-performance liquid chromatography. 298 6

In an attempt to determine the metabolic defect causing severe combined immunodeficiency (SCID) in horses in which altered purine metabolism has been observed, various parameters of purine and pyrimidine metabolism were evaluated. The activities of nine purine enzymes (adenosine kinase, purine nucleoside phosphorylase, deoxyadenosine kinase, deoxycytidine kinase, 5'-nucleotidase, AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase were measured in fibroblasts. All activities determined for SCID horses were normal. Uptake of 10 microM adenosine or 2'-deoxyadenosine (a growth inhibitory concentration for SCID fibroblasts) by SCID fibroblasts was identical to that found for normal fibroblasts in the presence of both 1 and 50 microM phosphate. The Km determined for the transport of both adenosine and 2'-deoxyadenosine was 35 microM. In the presence of p-nitrobenzylthioguanosine (a nucleoside transport inhibitor), 2'-deoxyadenosine uptake was inhibited to the same extent in all fibroblast lines tested. To determine if the last step in pyrimidine biosynthesis might be altered in SCID fibroblasts, UMP synthase activities were evaluated but found to be normal (0.5 nmol UMP formed/min/mg protein).
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PMID:Metabolic investigations of fibroblasts from horses, Equus caballus, with hereditary severe combined immunodeficiency. 299 78

The kinetic properties of highly purified human placental cytoplasmic 5'-nucleotidase were investigated. Initial velocity studies gave Michaelis constants for AMP, IMP, and CMP of 18, 30, and 2.2 microM, respectively. The enzyme shows the following relative Vmax values: CMP greater than UMP greater than dUMP greater than GMP greater than AMP greater than dCMP greater than IMP. The activity was magnesium-dependent, and this cation binds sequentially with a Km of 14 microM for AMP and an apparent Km of 6 mM for magnesium. A large variety of purine, pyrimidine, and pyridine compounds exert an inhibitory effect on enzyme activity. IMP, GMP, and NADH produce almost 100% inhibition at 1.0 mM. Nucleoside di- and triphosphates are potent inhibitors. ATP and ADP are competitive inhibitors with respect to AMP and IMP as substrates with Ki values of 100 and 15 microM, respectively. Inorganic phosphate is a noncompetitive inhibitor with Ki values of 19 and 43 mM. Nucleosides and other compounds studied produce only a modest decrease of enzyme activity at 1 mM. Our findings suggest that the enzyme is regulated under physiological conditions by the concentrations of magnesium, nucleoside 5'-monophosphates, and nucleoside di- and triphosphates. The nucleotide pool concentration regulates the enzyme possibly by a mechanism of heterogeneous metabolic pool inhibition. These properties of human placental cytoplasmic 5'-nucleotidase may be related to the control of nucleotide degradation in vivo.
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PMID:Human placental cytoplasmic 5'-nucleotidase. Kinetic properties and inhibition. 300 Oct 58

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.
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PMID:The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5'-nucleotidase. 302 68

Corynebacterium glutamicum cells are industrially used for glutamate production. However, the waste that contains microbial cells, cellular debris, residual sugars, ammonia and metabolites seriously pollutes the environment. The cells are recovered and utilized for ribonucleotide production so that the pollution caused by the cells is eliminated. Nucleic acid is extracted from the cells and is hydrolyzed with nuclease P1 from Penicillium citrinum. The hydrolysate is fractionated with Dowex-50 and Dowex-1 into 5'-CMP, 5'-UMP, 5'-GMP and 5'-AMP. The products are characterized by electrophoresis, ultraviolet light absorbance, and 5'-nucleotidase.
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PMID:Utilization of glutamate accumulating bacterial cells for production of ribonucleotides. 303 25

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

Rat brain myelin showed substantial activity of 5'-nucleotidase. The specific activity in myelin was enriched two- to threefold over that in rat brain homogenates, and the total activity in myelin accounted for approximately 24% of the activity in the homogenates. The 5'-nucleotidase in the homogenates and in isolated myelin had optimum activity at pH 7.5--9.0, was stimulated by Mg2+ and Mn2+, and was inhibited by Co2+, Zn2+, EDTA, and EGTA. 5'-AMP, 5'-UMP, and 5'-CMP were the preferred substrates, and 5'-GMP was hydrolyzed at approximately one-half the rate of the other mononucleotides. The very low rates of cleavage of beta-glycerophosphate and 2'-AMP ruled out any significant contribution of nonspecific phosphatase to the observed 5'-nucleotidase activity in myelin. The 5'-nucleotidase was inhibited by concanavalin A and was protected by alpha-methyl-D-mannoside against inhibited by that lectin, suggesting that this enzyme in the CNS is a glycoprotein. It is concluded from these data, and from histochemical observations made in other laboratories, that the myelin sheath is one major locus of 5'-nucleotidase in the rat brain.
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PMID:5'-nucleotidase in rat brain myelin. 625 85

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

The fate of UDP formed during the galactosylation of added N-acetylglucosamine in Golgi vesicles isolated from rat liver using D2O-sucrose gradients has been determined. UDP-Gal labeled with [14C]uracil was used, and the products of the reaction were separated and quantitated by using high-pressure liquid chromatography. [14C]Uridine rather than [14C]UDP or [14C]UMP was found to accumulate, indicating the presence of both UDPase and UMPase activities in the Golgi. Golgi vesicles were shown to contain a nucleosidediphosphatase activity that is membrane bound. It appears to be located on the luminal face of the Golgi since it is activated 3-5-fold by detergents and 4-fold by treatment of the vesicles with Filipin. We have shown previously that Filipin disrupts the Golgi but does not solubilize membrane-bound enzymes. The nucleosidediphosphatase of the Golgi differs from that present in rough endoplasmic reticulum in its absolute requirement for Ca2+ for activity and in its substrate specificity that is higher for UDP than for IDP. Golgi vesicles also contain UMPase activity that is stimulated only 2-fold by detergents or Filipin. Concanavalin A inhibits this activity about 80% in both intact and detergent-treated vesicles. The Golgi UMPase is thus probably identical with 5'-nucleotidase. These results are consistent with histochemical evidence from other laboratories that indicate that 5'-nucleotidase is present on both sides of liver Golgi membranes. In the presence of concanavalin A and N-acetylglucosamine, intact Golgi vesicles were found to convert UDP-Gal to UMP. These findings indicate that UDP formed by galactosyltransferase in the lumen of the vesicles is rapidly converted to UMP by UDPase in the lumen but that UMP moves rapidly out of the lumen of the Golgi and is broken down to uridine by 5'-nucleotidase on the cytoplasmic side of the vesicles.
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PMID:Orientation and role of nucleosidediphosphatase and 5'-nucleotidase in Golgi vesicles from rat liver. 629 86

Human red cell pyrimidine-5'-nucleotidase (EC 3.1.3.5) was partially purified from the blood of normal subjects by ion-exchange and affinity chromatography. Red cells were lysed in 50 mmol/l Tris-Cl buffer at pH 7.5 containing 1.0 mmol/l dithiothreitol and 0.5 mmol/l EDTA. The lysate was centrifuged and introduced onto a column of Sephadex A-50. After washing, the pyrimidine-5'-nucleotidase activity was eluted from the column with a NaCl gradient from 0 to 200 mmol/l in Tris buffer at pH 7.5. The pyrimidine-5'-nucleotidase was then desalted on Sephadex G-25 and introduced onto a UDP agarose column with a Tris buffer at pH 6.5 containing 150 mmol/l NaCl. This partial purification resulted in an approximately 80,000-fold increase in enzyme concentration. The Km for the partially purified enzyme was 0.32 mmol/l for UMP, 0.16 mmol/l for CMP and 0.11 mmol/l for OMP with a pH maximum of 7.5. This partially purified pyrimidine-5'-nucleotidase was then dialyzed in 50 mmol/l Tris-Cl buffer at pH 7.5 with 0.01 mmol/l CaCl2 and NaCl against 2 X 10(-3) mol/l 1,10-phenanthroline for 24 h at 4 degrees C. This incubation resulted in 73% decrease in enzyme activity which could be restored by the addition of zinc into the mixture, but not by the addition of other divalent metal ions.
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PMID:Partial purification and zinc dependence of human red cell pyrimidine-5'-nucleotidase. 631 32


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