3-Nucleotidase

5 -Nucleotidase (EC 3.1,3.5 5 -ribonucleotide phosphohy-drolase NTP) is a phosphatase that acts only on nucleoside-5 -phosphates, such as adenosine-5 -phosphate (AMP) and adenyhc acid, releasing inorganic phosphate. 

NTP is a glycoprotein widely distributed throughout the tissues of the body and is principally locaUzed in the cytoplasmic membrane of the cells in which it occurs. Its pH optimum is between 6.6 and 7.0.  

Despite its ubiquitous distribution, serum NTP activities appear to reflect hepatobiliary disease with considerable specificity. NTP is increased threefold to sixfold in those hepatobiliary diseases in which there is interference with the secretion of the bile. This may be due to extrahepatic causes (a stone or tumor occluding the bile duct), or it may arise from intrahepatic conditions, such as cholestasis caused by chlorpromazine, malignant infiltration of the liver, or bihary cirrhosis. When parenchymal cell damage is predominant, as in infectious hepatitis, serum NTP activity is only moderately elevated. 

Assay of NTP activity has been considered of value as an addition to measurement of nonspecific total ALP in patients with suspected hepatobiliary disease, and abnormal NTP activity is routinely interpreted as evidence of a hepatic origin of increased ALP activity in serum. However, approximately half of individuals in whom liver ALP activity is increased in serum may simultaneously show a normal NTP. On the other hand, increased NTP in the serum of patients with normal liver ALP is very often associated widi the presence of liver disease. Thus the frequent dissociation of the two enzyme activities supports the utility of determining both (fiver) ALP and NTP to increase the diagnostic efficiency for diseases of the liver. 

The substrates most generally used in measuring the activity of NTP are AMP or IMP (inosine-5 -phosphate). However, these substrates are organic phosphate esters and thus can be hydrolyzed to an appreciable degree by other nonspecific (alkaline) phosphatases, even at a pH as low as 7.5, which is the pH assumed optimal for NTP activity. Methods for the estimation of NTP in serum must therefore incorporate some means for correcting for the hydrolysis of the substrate by the nonspecific phosphatases. 

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 reaction mixture contained Tris-HCl-buffered UMP (purified free of uridine by ion-exchange chromatography) as the substrate and MgCl2. The reaction was started by the addition of the enzyme, and the incubation was carried out at 37°C for 60 minutes. The reaction was terminated by placing the reaction tubes in a boiling water bath for 3 minutes. After dilution and centrifugation, the supernatant solution was analyzed by HPLC. 

Erythrocytes from normal subjects were collected, washed, and lysed by dilution in distilled water. This lysate solution was used directly as the source of the enzyme. 

In the assay described by Amici et al. (1994), a wide variety of pyrimidine and purine nucleoside 5 -monophosphates were separated from their nucleosides by chromatography on a Supelco LClg guard column (4.6 mm x 20 mm, 5 fim). The short column allows separations in less than a minute. The mobile phase was 0.1 M potassium phosphate buffer (pH 6.0) except in the case of adenosine and deoxyadenosine, when 5% methanol was also included. The flow rate was 2 mL/min. Compounds were detected by monitoring the effluent at 254 nm, although sensitivity could be improved in some cases by using a different wavelength. 

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Enzymes, measured in clinical laboratories, for which kits are available include y-glutamyl transferase (GGT), alanine transferase [9000-86-6] (ALT), aldolase, a-amylase [9000-90-2] aspartate aminotransferase [9000-97-9], creatine kinase and its isoenzymes, galactose-l-phosphate uridyl transferase, Hpase, malate dehydrogenase [9001 -64-3], 5 -nucleotidase, phosphohexose isomerase, and pymvate kinase [9001-59-6]. One example is the measurement of aspartate aminotransferase, where the reaction is followed by monitoring the loss of NADH ... 

CarbocycHc 2/3 -didehydro-2/3 -dideoxyguanosine [118353-05-2] (carbovk, CBV, 66), C H 2N502, synthesized in 1988 (177), is a promising candidate for the chemotherapy of AIDS. CBV inhibits HIV repHcation and HIV-induced cytopathic effects in a variety of human T-lymphoblastoid ceU lines at concentrations approximately two hundred- to four hundredfold below its cytotoxic concentrations (177). CBV is as effective as AZT and DDC in reducing the expression of vkal antigen in HIV-infected CEM ceUs (177). The antivkal potency and selectivity of carbovk is comparable to the anti-HIV-1 potency and selectivity of 2/3 -dideoxyadenosine (178). The exact mode of antivkal action of carbovk has not yet been elucidated, but may be the modulating effect of intraceUular nucleotides on 5 -nucleotidase activity (179). 

As for the stingray venom, not much is known. There was a report on the presence of 5 -nucleotidase and phosphodiesterase in stingray, Urolophus halleri, venom (29). 

Ribonucleoside 5 -0-hydroxymethylphosphonates (8 R = OH) are resistant to the action of phosphatases and phosphodiesterases. They are, however, good substrates for snake venom 5 -nucleotidase, unlike (8 R = H).2 ... 

Pyrimidine 5 -nucleotidase (P5N) is a unique enzyme that was recognized from studies of families with relatively common hemolytic disorders. The enzyme catalyzes the hydrolytic dephosphorylation of pyrimidine 5 -nucleotides but not purine nucleotides. The role of this enzyme is to eliminate RNA and DNA degradation products from the cytosol during erythroid maturation by conversion of nucleotide monophosphates to diffusible nucleosides. P5N is inhibited by lead, and its activity is considered to be a good indicator of lead exposure (PI). 

Fig. 6. Basophilic stippling in peripheral blood smear of a patient with pyrimidine 5 -nucleotidase deficiency.

Pyrimidine 5 -nucleotidase (P5N) deficiency appears to be the third most common cause of hereditary nonspherocytic hemolytic anemia after G6PD and PK deficiencies. To date, more than 42 cases have been reported worldwide (FI 1) since the first report by Valentine et al. (V4). This syndrome is characterized by hemolytic anemia, pronounced basophilic stippling of red blood cells (Fig. 6), and a... 

H6. Hirono, A., Fujii, H Natori, H., Kurokawa, I., and Miwa, S., Chromatographic analysis of human erythrocyte pyrimidine 5 -nucleotidase from five patients with primidine 5 -nucleotidase deficiency. Br. J. Haematol. 65,35-41 (1987). 

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). 

V4. Valentine, W. N., Fink, K Paglia, D. E., Harris, S. R., and Adams, W. S., Hereditary hemolytic anemia with human erythrocyte pyrimidine 5 -nucleotidase deficiency. J. Clin. Invest. 54, 866-879 (1974). 

ALA = 5-aminolevulinic acid ALAD = 6-aminolevulinic acid dehydratase ALAS = 5-aminolevulinic acid synthase EP = erythrocyte protoporphyrins FEP = free erythrocyte protoporphyrins FSH = follicle stimulating hormone IQ = intelligence quotient LH = luteinizing hormone NS = not specified (occup) = occupational Py-5 -N = pyrimidine-5-nucleotidase TSH = thyroid stimulating hormone ZPP = erythrocyte protoporphyrin... 

The increase in erythrocyte destruction may be due in part to inhibition by lead of pyrimidine-5 -nucleotidase, which results in an accumulation of pyrimidine nucleotides (cytidine and uridine phosphates) in the erythrocyte or reticulocyte. This enzyme inhibition and nucleotide accumulation affect erythrocyte membrane stability and survival by alteration of cellular energetic (Angle et al. 1982 EPA 1986a). Formation of the heme-containing cytochromes is inhibited in animals treated intraperitoneally or orally... 

Buc HA, Kaplan JC. 1978. Red-cell pyrimidine 5 -nucleotidase and lead poisoning. Clin Chim Acta 87 49-55. 

Paglia DE, Valentine WN, Dahigren JG. 1975. Effects of low-level lead exposure on pyrimidine 5 -nucleotidase and other erythrocyte enzymes Possible role of pyrimidine 5 -nucleotidase in the pathogenesis of lead-induced anemia. J Clin Invest 56 1164-1169. 

Alanko, L., Heiskanen, S., Stenberg, D. Porkka-Heiskanen, T. (2003a). Adenosine kinase and 5 -nucleotidase activity after prolonged wakefulness in the cortex and the basal forebrain of rat. Neurochem. Int. 42 (6), 449-54. 

The biosynthesis of adenosine is theoretically controlled by several processes namely (1) the biosynthesis of adenosine from AMP by 5 -nucleotidase [EC 3.1.3.5], (2) from S-adenosyl homocysteine by S-adenosyl homocystine hydrolase [EC 3.3.1.1], (3) the metabolism of adenosine to AMP by adenosine kinase [EC 2.7.1.20], and (4) to inosine by adenosine deaminase (ADA) [EC 3.5.4.2], Interestingly, both 5 -nucleotidase and ADA activities were found to be highest in the leptomeninges of rat brain in contrast, the adenosine kinase activity was widely distributed throughout the brain parenchyma, which has negligible ADA activity... 

The bearing which these discoveries have had on the elucidation of the structure of ribopolynucleotides will be discussed later. It is important to stress here, however, that, for most purposes, the older methods of preparing nucleotides have been superseded by procedures which yield separate isomers of each. Of the techniques mentioned above, paper chromatography iB mainly of analytical value, and is the most convenient method for the qualitative detection of isomeric adenylic acids. The only disadvantage of this method is that the isomers are not completely separable from muscle adenylic acid. The presence of the latter, however, can be readily detected by hydrolyzing it to adenosine by means of the specific 5-nucleotidase present in snake venoms,66 or by deamination by a specific enzyme... 

Certain enzymes shown to be present in myelin could be involved in ion transport. Carbonic anhydrase has generally been considered a soluble enzyme and a glial marker but myelin accounts for a large part of the membrane-bound form in brain. This enzyme may play a role in removal of carbonic acid from metabolically active axons. The enzymes 5 -nucleotidase and Na+, K+-ATPase have long been considered specific markers for plasma membranes and are found in myelin at low levels. The 5 -nucleotidase activity may be related to a transport mechanism for adenosine, and Na+, K+-ATPase could well be involved in transport of monovalent cations. The presence of these enzymes suggests that myelin may have an active role in ion transport in and out of the axon. In connection with this hypothesis, it is of interest that the PLP gene family may have evolved from a pore-forming polypeptide [9],... 

ATP diphosphohydrolase Diadenosine polyphosphatase 5 nucleotidase Nucleoside transporter Adenosine deaminase Adenosine kinase Xanthine oxidase Nucleoside phosphorylase... 

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