5 -Nucleotidase assay

M26. Miwa, S., Luzzatto, L., Rosa, R., Paglia, D. E., Schroter, W De Flora, A., Fujii, H., Board, P. G and Beutler, E., International Committee for Standardization in Haematology Recommended methods for an additional red cell enzyme (pyrimidine 5 -nucleotidase) assay and the determination of red cell adenosine 5 -triphosphate, 2,3-diphosphoglycerate and reduced glutathione. Clin. Lab. Haematol. 11, 131-138 (1989). 

A slope of increasing fluorescent intensity can be used to quantitate virus particle (or amplified gene) quantities in 5 -nucleotidase assays. 

Figure 9.89 Pyrimidine 5 -nucleotidase assay in undialyzed human erythrocyte lysate obtained by 1 5 dilution of packed cells with deionized water, (a) Separation of 1 nmol of CMP, cytidine, and uridine as the standards. Separation of the assay mixture, containing 50 mM Tris-HCl, pH 7.5, 0.2 mM CMP, 1 mM MgCl2, 1 mM DTT, and 20 fiL of lysate in 0.5 mL of total volume (b) at time zero and (c) after 30 minutes of incubation (c). (From Amici et al., 1994.)...

Adenosine S -monophosphate (AMP) is used as a substrate for S -nucleotidase assay. However this substrate can also be hydrolysed by nonspecific phosphatases. Nickel ions inhibit S -nucleotidase but not the nonspecific phosphatases. Serum is therefore incubated with AMP, with and without nickel ions and the amounts of inorganic phosphate liberated by the reactions are measured. The difference between the two values corresponds to the activity of 5 NT. 

Brydon and Roberts- added hemolyzed blood to unhemolyzed plasma, analyzed the specimens for a variety of constituents and then compared the values with those in the unhemolyzed plasma (B28). The following procedures were considered unaffected by hemolysis (up to 1 g/100 ml hemoglobin) urea (diacetyl monoxime) carbon dioxide content (phe-nolphthalein complex) iron binding capacity cholesterol (ferric chloride) creatinine (alkaline picrate) uric acid (phosphotungstate reduction) alkaline phosphatase (4-nitrophenyl phosphate) 5 -nucleotidase (adenosine monophosphate-nickel) and tartrate-labile acid phosphatase (phenyl phosphate). In Table 2 are shown those assays where increases were observed. The hemolysis used in these studies was equivalent to that produced by the breakdown of about 15 X 10 erythrocytes. In the bromocresol green albumin method it has been reported that for every 100 mg of hemoglobin/100 ml serum, the apparent albumin concentration is increased by 100 mg/100 ml (D12). Hemolysis releases some amino acids, such as histidine, into the plasma (Alb). 

Serum ALP and total bilirubin (unconjugated and conjugated fractions) are traditionally used to monitor cholestatic injury. The ALP families of enzymes are zinc metalloproteases that are present in nearly all tissues. In the liver, ALP is immu-nolocalized to the microvili of the bile canaliculus [124]. Increased synthesis of ALP and its release into the circulation occurs within hours of cholestatic injury [129]. Serum assays of 5 -nucleotidase (5 -NT) or y-glutamyltransferase activity (GGT) are used to confirm the liver as the specific origin for the elevation of ALP. Increases in serum bilirubin or bile acids are usually the result of bile retention subsequent to impaired bile flow, increased production associated with accelerated erythrocyte destruction, or altered bilirubin metabolism [129]. 

Fig. 5. Illustration of the 5 -nucleotidase (TaqMan) assay for allele discrimination. (A) The allele discrimination assay employs two unlabeled PCR primers and two doubly fluorescent labeled PCR probes for visuaUzation of a mutant allele. The target sequence is initially denatured and amplified in the presence of each of the primers and probes. Increasing polymerization in the presence of a thermostable polymerase which contains a 5 proofreading function allows cleavage of one fluorescent indicator from an appropriate probe during the cycling reaction. (B) Probes are designed with a fluorescent reporter and a quencher moiety. AmpUfication reactions are spiked with additional fluorescent quenchers in order to render the reaction initially dark to the photomultipUer mbe or diode. The probes are designed...

We discuss two assays for the measurement of pyrimidine 5 -nucleotidase activity. In the first, as described by Sakai et al. (1982), a pyrimidine nucleoside 5 -phosphate is hydrolyzed to form the corresponding pyrimidine nucleoside. 

The activity is found in erythrocytes, platelets, and lymphocytes, and determination of its value aids in diagnosis of some blood disorders. In this assay, which can readily be used for purine and pyrimidine 5 - and 3 -nucleotidase activities, the nucleoside monophosphate (the substrate) was separated from the nucleoside (the product) using ion-pair reversed-phase HPLC with a mobile phase of 5% methanol-5 mAf potassium dihydrogen phosphate 0.25 mAf 1-decanesulfonic acid was also added to the mobile phase. The elution was carried out at room temperature and the eluent monitored at 254 nm. 

The enzyme 5 -nucleotidase dephosphorylates IMP to inosine and P. Thus, since this reaction represents a possible fate for the IMP formed by the transferase (Fig. 10.7), reconstitution studies were undertaken with the nucleotidase. These studies were carried out using the HPLC assay method developed for the HGPRTase activity. A reaction mixture was prepared that contained hypoxanthine and PRibPP as substrates. The reaction was started by the addition of purified HGPRTase enzyme. Samples were removed and were analyzed by HPLC. The chromatographic profiles obtained at 0,10, 20, and... 

By using highly specific radioactive cyclic nucleotides and the snake venom nucleotidase, the most sensitive, specific and convenient method of assay for phosphodiesterase has been evolved. 

The assay is carried out both at 1 jaM and 1 mM substrate concentrations [162]. The enzyme preparation is incubated with tritium-labelled cyclic nucleotide in the presence of Mg, the reaction is stopped by brief heating in a water bath, the 5 -nucleotide formed is converted to the nucleoside by snake venom 5 -nucleotidase, the nucleoside is separated from the remaining cyclic nucleotide by anion exchange chromatography (Dowex 2), and the radioactivity of both compounds is counted. 

The entire assay can conveniently be carried out in a glass scintillation vial [82]. The reaction volume contains Tris buffer (pH 7.5), Mg, [ H]cyclic nucleotide, and snake venom 5 -nucleotidase. After a 10-min incubation at 37°C, the reaction is stopped by the addition of a slurry of AG1-X2 anion exchange resin. The resin binds substrate cyclic nucleotides but does not bind nucleosides. Scintillation fluid is added and the amount... 

An approximate idea of the distribution of acid phosphatase activity in human tissues, regardless of the nature of the acid phosphatase, may be obtained from the studies of Reis (R2) on 5 -nucleotidase and other phosphomonoesterases. He prepared aqeuous homogenates of postmortem tissue in the proportion of 20 parts of water to one of tissue, allowed these to autolyze for 2 days at room temperature, centrifuged the material, and employed the supernatant fluid. The assay mixture consisted of 0.4 ml of a suitable buffer, 0.1 ml of 0.005 Af phenyl phosphate as substrate, and 0.1 ml of tissue extract. The enzyme activity was expressed as micrograms of phosphorus hydrolyzed per hour per milligram of wet... 

Zinc-Dependent Enzymes. Despite the large number of zinc metaUoenzymes that have been identified, no single enzyme assay has yet found acceptance as an indicator of zinc status. This may be due to avid retention of zinc by these enzymes, even in the face of dietary zinc depletion, and to difficulties with reproducible measurements of activity. However, bone-specific alkaline phosphatase, extracellular SOD, lymphocytes, and plasma 5-nucleotidase appear to be responsive to zinc intake. ... 

Two publications on the higher ketose mono- and bis-phosphates in rat-liver extracts have described their detection and estimation by colorimetry and enzymic assay,and the synthesis of octulose 1,8-bisphosphates using muscle aldolase. A stereospecific synthesis of adenosine 3, 5 -cyclic phosphothioate has appeared/ Standard condensation methods have been used to synthesize the 5 -O-(Taminoethane-phosphonyl) nucleosides (21) and (22). The compounds were shown to be inert to the action of alkaline phosphatase and are poor substrates for 5 -nucleotidase. The selenophosphates (23) and (24) have been prepared.Phosphorylation of nucleosides using dibenzyl hydrogen phosphate,... 

Fibroblasts were harvested by brief incubation at 37 C in 0.05 % trypsin in PBS. Cells were collected by centrifugation and washed with 0.154 M NaCl. Extracts were prepared by 4 cycles of freezing and thawing. Samples of the supernatant after centrifugation were used for HGPRT or APRT activity, and assays of protein content. The enzyme activities were determined in the presence of 100 juM a, B-methylene-adenosine-5 -diphosphate an inhibitor of 5 -nucleotidase as described by Hershfield et al. ... 

Recent results (see Bontemps et al., this volume) have shown that, contrary to our previous conclusion, the cytoplasmic 5 -nucleotidase operates in hepatocytes in basal conditions and participates in a futile cycle. This activity could be confirmed when a highly purified cytoplasmic 5 -nucleotidase, prepared according to Itoh (1981) was assayed in physiological conditions (see also Fig. 4) instead of the partially purified enzyme we used previously. 

Nucleotidase activity was measured in human erythrocyte lysates which had been dialysed overnight against Tris/HCl buffer, pH 8.0 containing 20 mM mercaptoethanol and then diluted 1 5 in the same buffer. The assay mixture contained 0.5 ml haemolysate, 0.2 ml 0.1 M MgCl and 0.2 ml 0.04 M 5 ribonucleotide substrate in 0.05 M Tris/HCl buffer pH 8.0 and was incubated at 37°C for 1-2 hrs before adding 0.5 ml 20% trichloroacetic acid. Phosphate determination was carried out on the clear supernatant using the Chen modification (6) of the Fiske-SubbaRow procedure. 

The assay data are summarized in Table 2. The patient s erythrocytes show marked deficiency of pyrimidine nucleotidase activity with CMP and UMP but normal levels of activity with dUMP and dTMP as substrates. 

The results obtained by electrophoresis and quantitative assay are complementary and support the hypothesis that there are two types of pyrimidine nucleotidase in human erythrocytes. The patient shows deficient activity with UMP, CMP and dCMP, the preferred substrates of rodent UMPH-1 and this agrees with the conclusion that rodent UMPH-1 and the human pyrimidine-specific nucleotidase identified by Valentine et al. (1) and purified by Torrance et al. (3) are homologous. 

The loss of HGPRT and APRT activities by freezing and thawing of the cells has been dealt with previously and can be prevented by the use of concentrated cell suspensions and the addition of PRPP (1). The presence of nucleotidase activity in the cell extracts causes the rapid degradation of the nucleotides, produced in the assay, to their corresponding nucleosides. 

It is noteworthy that for the Lesch-Nyhan syndrome fibroblasts, the assay in the presence of TTP furnished the bulk of the radioactivity in the nucleoside fraction rather than in the nucleotide fraction, in contrast to normal cell extracts in which the bulk of the radioactivity is found in the nucleotide fraction. Two possible explanations may be entertained to explain this observation. First, in the HGPRT-deficient system the nucleotidase activity is far in excess over the HGPRT activity, and presumably the residual nucleotidase activity, not inhibited by TTP, is sufficient to degrade the relatively small amount of nucleotide formed. Secondly, the possibility should be considered that the accumulation of radioactivity in the nucleoside fraction reflects the anabolic activity of nucleoside phospho-rylase reacting the purine base with ribose-l-phosphate to form the nucleotide by an alternative pathway. However, this latter explanation seems to be invalid in view of the absence of the suitable substrate, ribose-l-phosphate, from the incubation system, and by the linearity... 

ATP inhibit nonspecific nucleotidases. Another way to prepare [a P] GTP Arfl for the GAP assay is to incubate Arfl with 25 ruM HEPES, pH 7.4, 100 mM NaCl, 3.5 mM MgCl2, 1 mM EDTA, 1 mM ATP, 1 fjM [a PJGTP (specific activity = 50,000-250,000 cpm/pmol), 25 ruM KCl, 1.25 U/ml pyruvate kinase, and 3 mM phosphoenolpyruvate. This buffer contains a GTP regenerating system. If using Arfl that has not been myristoy-lated, include 0.1% (w/v) Triton X-100. For myristoylated Arfl, use either micelles of 3 mM dimyristoylphosphatidylcholine and 0.1% cholate, pH 7.4 or use vesicles prepared by extrusion or sonication (see Chapter 15 of this volume. Assay and Properties of the Arf GAPs AGAPl, ASAPl, and ArfGAPl). 

Several sources of background counts are present in this assay and need to be considered. One is the free Pi in the commercially obtained isotope preparation. This background steadily increases as the nucleotide preparation ages due to spontaneous hydrolysis of the GTP and radiation damage. Another is caused by the presence of nucleotidases and nucleoside... 

Nucieotidases.—Since the 5 -nucleotidase from Dictyostelium discoideum interacted with immobilized concanavalin A, it appears to be a glycoprotein. Pectinesterases.—The occurrence, formation, assay, purification, and specificities of pectinesterases have been reviewed. Pectinesterase activity was detected both in healthy and diseased onions Allium cepa), but not in cultures of the invading bacterium (Pseudomonas cepacia) Both the pectinesterase and poly-D-galact-uronate lyase activities of Clostridium multifermentans are associated with a single complex. ... 

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