Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P47989 (xanthine oxidase)
 8,633 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using density gradient centrifugation, human trophoblastic cells were enriched from mixed cell populations of enzymatically dispersed first- and third-trimester placentae. Over 95 per cent of the cells recovered were of epithelial (i.e., trophoblastic) origin, as evidenced by their cytokeratin intermediate filament positivity and vimentin negativity, examined using indirect immunofluorescence, and also by their high content of human chorionic gonadotrophin. The activities of key enzymes involved in purine degradation and re-utilization (5'-nucleotidase; AMP-deaminase; hypoxanthine phosphoribosyltransferase (HPRT); xanthine dehydrogenase/oxidase) as well as the total activity of alkaline phosphatase were measured in the trophoblastic cells. A six-fold increase in the trophoblastic alkaline phosphatase activity was noted between the first and third trimester. A 40 per cent decrease was noted in the activity of 5'-nucleotidase, which, on the basis of kinetic properties, appears to have a dominant role in the dephosphorylation of placental nucleoside-5'-monophosphates. The trophoblastic activities of AMP-deaminase, HPRT, and xanthine dehydrogenase/oxidase did not change as a function of the gestational age. In view of the relative activities of the latter two enzymes, hypoxanthine formed in the trophoblast appears more likely to be re-utilized than degraded to uric acid.
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PMID:Activities of key enzymes of purine degradation and re-utilization in human trophoblastic cells. 283 9

The enzymatic inosine 5'-monophosphate assay described by Grassl [in, Methods of Enzymatic Analysis (H. U. Bergman, ed.), pp. 2168-2171, Academic Press, New York (1974)] is highly nonspecific, as ITP, ATP, ADP, AMP, and adenosine react stoichiometrically. The reactivity with the adenine derivatives is due to the tri- and diphosphatase activity of alkaline phosphatase (AP), coupled with adenosine deaminase (and possibly AMP deaminase) contamination of commercially available preparations of AP, purine-nucleoside phosphorylase, and/or xanthine oxidase. The inclusion of coformycin (0.05 microgram/ml), a potent inhibitor of these deaminases, completely eliminated the cross-reactivity. ITP, however, still reacted stoichiometrically due to the tri- and diphosphatase activity of AP. Meyer and Terjung [Amer. J. Physiol. 237 C111-C118 (1979)] introduced a modification of Grassl's procedure, substituting 5'-nucleotidase for AP. It has been found that this disallows reactivity with ATP, ADP, and ITP but that AMP and adenosine still react completely. Coformycin prevents this cross-reactivity. It is therefore recommended that the assay be carried out with 5'-nucleotidase (instead of AP) and coformycin, in order to achieve a more specific assay, and one more suitable for use with whole tissue extracts.
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PMID:An enzymatic inosine 5'-monophosphate assay of increased specificity. 298 81

Coupled enzyme assays are described for measuring inorganic phosphates, organic phosphates and phosphate-liberating enzymes in biological material. The assays all determine Pi by its reaction with inosine, catalysed by nucleoside phosphorylase; this yields ribose 1-phosphate and hypoxanthine. The hypoxanthine is oxidized to uric acid by xanthine oxidase, and may be measured either by the absorbance of the uric acid, or by the formazan formed when a tetrazolium salt is used as the oxidant. The coupled enzyme assays are characterized by high sensitivity, quantitative utilization of phosphates and stoichiometric formation of the measurable products, measurement at pH 6.0-8.5, determination of phosphates within a single analytical step, and continuous measurement of phosphohydrolase activity in a corresponding rate assay. Examples include determinations of substrates such as Pi, PPi and AMP, and of enzymes such as 5'-nucleotidase, inorganic pyrophosphatase and glucose-6-phosphatase. Directions for further examples are given.
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PMID:Enzymic determination of inorganic phosphates, organic phosphates and phosphate-liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions. 299 93

A simple method is presented for the determination of pyrimidine-5'-nucleotidase activity using a continuous spectrophotometric assay system. Activity is determined by measuring inorganic phosphate generation using a linked indicator system that produces uric acid in the presence of inosine, purine nucleoside phosphorylase, and xanthine oxidase. This method has several advantages over any of the methods currently in use.
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PMID:A continuous spectrophotometric assay for pyrimidine-5'-nucleotidase. 300 35

1. Proteins of fat-globule membrane from bovine milk were solubilized with the non-ionic detergent Triton X-100 in the presence of protease inhibitors. Approximately 25% of the total membrane protein was solubilized and the extracts were shown to contain a sample of most of the major membrane proteins and glycoproteins. 2. The solubilized proteins were separated in flat-beds of Ultrodex by electrofocusing and the pI values for the major proteins, glycoproteins and certain enzymes determined. Several of the proteins displayed marked heterogeneity indicating the existence of protein variants and isoenzymes. Principal pI values for the enzymes assayed were as follows: xanthine oxidase, 7.35--7.55; NADH2: iodonitrotetrazolium reductase, less than 4.5; 5'-nucleotidase, 7.15--7.4; alkaline phosphatase, 5.4--5.7; phosphodiesterase, 4.6--4.8; gamma-glutamyl transpeptidase, 4.4--4.55. 3. Fractions after electrofocusing were analyzed by 'fused rocket' immunoelectrophoresis and crossed immunoelectrophoresis after separation in polyacrylamide gels containing sodium dodecyl sulphate. Major antigens of the membrane include xanthine oxidase and glycoproteins of apparent molecular weights 67 000, 49 500 and 46 000. The latter two components share common antigenic determinants and could not be separated by gel filtration, ion-exchange chromatography, lectin-affinity chromatography or preparative electrofocusing.
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PMID:Separation of the proteins of bovine milk-fat-globule membrane by electrofocusing with retention of enzymatic and immunological activity. 610 13

Crude tissue or tumor extracts either do not contain sufficient inosine 5'-monophosphate dehydrogenase (IMPD) activity to be measured spectrophotometrically, or interfering enzyme activities prevent the use of a more sensitive radiochemical assay. A modified assay system which incorporates alpha, beta-methylene adenosine 5'-diphosphate, an inhibitor of 5'-nucleotidase; allopurinol, an inhibitor of xanthine oxidase; and ethylenediaminetetraacetate, an inhibitor of alkaline phosphatase, has been developed. [14C]Xanthine monophosphate produced during the assay was separated from [14C]hypoxanthine monophosphate by thin-layer chromatography on flexible diethylaminoethyl-cellulose sheets. Xanthine monophosphate formation was linear for at least 40 min and was inhibited by greater than 95% in the presence of mycophenolic acid, a specific IMPD inhibitor. Partial purified IMPD from murine EMT6 tumors was used to compare assay rates obtained with the radiochemical and spectrophotometric assays under identical conditions. The reaction rate of the radiochemical assay was 0.92 +/- 0.07 (S.E.) of the rate of xanthine monophosphate formation as determined spectrophotometrically at 290 nm, indicating that both assays are measuring product formation with an equal degree of accuracy. The improved radiochemical assay was used to determine IMPD specific activity in supernatants from EMT6 tumors and several normal mouse tissues. The observed activities (nmol/min/mg protein) were: EMT6 tumor, 0.303; spleen, 0.029; brain, 0.022; kidney, 0.015; lung, 0.009; liver, 0.008; and heart and skeletal muscle, less than 0.004.
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PMID:Sensitive radiochemical assay for inosine 5'-monophosphate dehydrogenase and determination of activity in murine tumor and tissue extracts. 613 40

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

Adenosine kinase, adenosine deaminase, hypoxanthine phosphoribosyltransferase, inosine-nucleoside phosphorylase, 5'-AMP deaminase and 5'-IMP nucleotidase were identified in cell-free extracts of duckling erythrocytes; no evidence for 5'-AMP nucleotidase and xanthine oxidase activity was found. The Km values for the duckling red cell enzymes were similar to those reported for human erythrocytes. Plasmodium lophurae extracts demonstrated similar enzyme activities except for 5'-AMP deaminase and 5'-IMP nucleotidase which were absent. It is proposed that during infection erythrocytic AMP is catabolized to IMP, inosine and hypoxanthine; the hypoxanthine is taken up by the plasmodium, utilized to form IMP, and this in turn is converted into adenine and guanine nucleotides.
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PMID:Purine metabolizing enzymes of Plasmodium lophurae and its host cell, the duckling (Anas domesticus) erythrocyte. 678 22

Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5'-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg2+-ATPase, (Na+ + K+)-ATPase, gamma-glutamyl transpeptidase, phosphodiesterase I, alkaline phosphatase and xanthine oxidase were also high (12-18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide composition in both light and heavy membranes were similar upon SDS-polyacrylamide gel electorphoresis.
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PMID:Isolation and characterization of plasma membrane from lactating bovine mammary gland. 720 55


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