Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.17.3.2 (xanthine oxidase)
 8,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A blotting method is described to detect enzymes that do not normally yield a colored product. The method can be used for dot blotting as well as blotting after gel electrophoresis of many enzymes if the reactions they catalyze can be coupled to an oxidase or a dehydrogenase. The latter, designated "auxiliary enzymes," are preimmobilized on membranes of nitrocellulose or positively charged nylon and the reaction they catalyze is coupled with reduction of tetrazolium salt to yield colored formazan on areas of the transfer membrane occupied by the blotted enzymes. In the examples reported here, preimmobilized glucose oxidase, L-amino acid oxidase, xanthine oxidase, malate dehydrogenase, and a mixture of hexokinase and glucose-6-phosphate dehydrogenase were used as auxiliary enzymes to detect blotted invertase, leucine aminopeptidase, purine nucleoside phosphorylase, fumarase, and adenylate kinase, respectively. Detection limits varied, but never exceeded 100 ng for these enzymes. After blotting from polyacrylamide gels, the fumarase assay was the most sensitive of those investigated, detecting 10 ng of enzyme used for electrophoresis. Invertase, a glycoprotein, was detected with higher sensitivity on nitrocellulose membranes when concanavalin A was present on the membrane in addition to the auxiliary enzyme, glucose oxidase. On blots from isoelectric focusing gels, the assay detected two isozymes of purine nucleoside phosphorylase in a sample from calf spleen and at least five isozymes of this enzyme in lysates from human red cells.
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PMID:Activity staining of blotted enzymes by reaction coupling with transfer membrane-immobilized auxiliary enzymes. 245 38

Reaction conditions for determining the activity of purine nucleoside phosphorylase (PNP; E.C. 2.4.2.1) were investigated. We examined the kinetic parameters for the enzymatic reaction with respect to the substrates inosine and phosphate. We confirmed the pH optimum, established the optimal concentration of xanthine oxidase and that of calcium and magnesium. The Km values for inosine and phosphate were found to be 60 uM and 667 uM, respectively. Optimum assay conditions for PNP activity were established. This optimized method has been compared with other procedures and found to be more sensitive and to yield significantly higher activities. The experimental variation of a manual procedure using these optimum reaction conditions was less than 4.5%. The mean erythrocyte PNP activity of 28 healthy subjects was estimated to be 9.71 U/mL packed cells at 25 degrees C and 18.60 U/mL packed cells at 37 degrees C.
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PMID:Purine nucleoside phosphorylase in erythrocytes: determination of optimum reaction conditions. 249 71

A new colorimetric procedure for the determination of purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) activity is described. In this procedure, the hydrogen peroxide formed in the PNP-xanthine oxidase reaction is used to oxidize the chromogenic reagents--3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone using the enzyme peroxidase. The rate of enzyme reaction is followed at 520 nm. This procedure correlated well with the UV method (290 nm), with a correlation coefficient of 0.98 (P less than 0.005). Within-run and between-run precision (CV) were less than 2.8% and 3.7%, respectively. Here we also describe an optimized NAD-dependent method (340 nm) for PNP determination. The colorimetric method is superior to both the 340 nm and the UV methods in terms of both sensitivity and precision. The mean erythrocyte PNP activity for 17 healthy subjects was 11.88 U/mL packed cells for the NAD-dependent method and 13.22 U/mL packed cells for the colorimetric method.
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PMID:A new colorimetric assay for purine nucleoside phosphorylase. 250 13

Probenecid decreased the plasma concentration of oxypurines (hypoxanthine and xanthine) but did not increase the renal excretion of oxypurines. However, the concentrations of hypoxanthine and nucleotides (inosine monophosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, guanosine diphosphate and guanosine triphosphate) in red blood cells did not change after the administration of probenecid. In addition, the drug did not inhibit adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine guanine phosphoribosyl transferase and xanthine oxidase in vitro. These results suggested that the rapid fall of plasma concentration of uric acid due to the potent uricosuric action of probenecid resulted in the fall of plasma concentration of oxypurines.
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PMID:Effect of probenecid on oxypurines in plasma. 251 Nov 59

A flow injection method with chemiluminescence detection has been developed for the enzymatic determination of inorganic phosphate. Purine nucleoside phosphorylase, xanthine oxidase, and urate oxidase were immobilized on controlled-pore glass beads. Hydrogen peroxide released by the enzymatic reactions of phosphate and inosine in carrier buffer was detected by the luminol-microperoxidase system in a flow cell. The calibration graph was linear over the range of 5 to 250 pmol, and reproducibility was 1.75% at 10 pmol. The detection limit was 500 fmol of phosphate in 50 microliters of sample injected. The phosphate content in deoxyribonucleic acid was measured by this method.
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PMID:Determination of inorganic phosphate by flow injection method with immobilized enzymes and chemiluminescence detection. 251 13

We have developed a new assay for purine nucleoside phosphorylase which is based on the release of tritium when [2-3H]inosine is used as the substrate and the reaction is coupled with xanthine oxidase. After the reaction is terminated, residual [2-3H]inosine is adsorbed on charcoal and the supernatant solution is assayed for radioactivity by liquid scintillation spectrometry. The new method gave results indistinguishable from those obtained by spectrophotometric determination of uric acid produced by the phosphorylase-xanthine oxidase-coupled reaction or by radioassay of chromatographically isolated [8-14C]hypoxanthine when [8-14C]inosine was used as substrate. The new method is faster than those involving chromatographic isolation of products. In comparison with spectrophotometric methods, it not only requires less manual time, but it also has the advantage that it can be used to study inhibitors whose ultraviolet absorption might interfere with spectrophotometric determination of uric acid.
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PMID:A new isotopic assay for purine nucleoside phosphorylase. 251 16

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

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

The performance of an enzymatic colorimetric method for the determination of inorganic phosphorus in serum and urine is described. Phosphate ions react with inosine in the presence of purine nucleoside phosphorylase to form hypoxanthine; this is oxidized by xanthine oxidase to uric acid with production of hydrogen peroxide. The latter is determined with the aid of the chromogen system peroxidase/4-aminophenazone/N-ethyl-N-(3-methylphenyl)-N'-acetylethyl enediamine , the coloured product being measured at 555 nm. This series of reactions is completed in 5 min at 37 degrees C. The test is linear up to 240 mg/l. Analytical recovery in serum averaged 101.2 +/- 1.2% and in urine 101.9 +/- 3.2%. Within-run and between-run precision studies in serum and urine samples gave CVs less than or equal to 4.54% (at 22.0 mg/l). Results obtained by this method agree (r = greater than or equal to 0.983) with the molybdate UV and molybdenum blue methods. Interference by endogenous substances, including organic phosphate, was negligible.
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PMID:Enzymatic colorimetric method for the determination of inorganic phosphorus in serum and urine. 313 8

Coformycin, which is an inhibitor of adenosine deaminase, significantly inhibited in vitro blastogenic responses of human lymphocytes to both phytohaemagglutinin (PHA) and pokeweed mitogen (PWM), whereas blastogenic responses to bacterial lipopolysaccharide (LPS) were rather enhanced by the addition of coformycin. Blastogenic responses of lymphocytes to PHA and PWM were markedly suppressed by the addition of adenosine, which is a substrate of adenosine deaminase. Allopurinol, which is an inhibitor of xanthine oxidase, inhibited blastogenic responses of human lymphocytes to PHA, PWM, and bacterial LPS. Inosine (a substrate of purine nucleoside phosphorylase) and hypoxanthine (a substrate of xanthine oxidase) showed no or only a small effect on blastogenic responses of human lymphocytes. These results suggest that adenosine deaminase activity is associated with the T-cell response but not with the B-cell response and that the impaired T-cell response in adenosine deaminase deficiency is the result of intracellular retention of adenosine in T cells. The results also suggest that purine nucleoside phosphorylase or xanthine oxidase activity is associated with both T- and B-cell responses.
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PMID:Purine metabolic enzymes in lymphocytes. IV. Effects of enzyme inhibitors and enzyme substrates on the blastogenic responses of human lymphocytes. 392 75


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