S -nucleotidase

Figure 2.10 The use of enzymes to identify an unknown compound. The compound tentatively identified as IMP, based on its retention time of 2 minutes (chromatogram obtained at zero reaction time), was incubated with a commercially available preparation of S -nucleotidase. Samples of the incubation mixture were removed and analyzed by HPLG The chromatograms, obtained at 10 and 20 minutes of reaction time, showed a reduction in the area of the IMP peak and an increase in the area of the inosine (Ino) peak, confirming that the original peak was IMP.

Figure 9.93 HPLC chromatograms of phosphomonoesterase hydrolysis of A(S)MP. (i4) Chromatogram obtained from calf intestinal mucosa alkaline phosphatase hydrolysis of A(S)MP. In a reaction volume of 100 /xL containing 100 mM Tris-HCl (pH 8.1), 300 pM A(S)MP, and 20 mM MgCl2, the reaction was initiated by addition of 2 of enzyme and incubated at 30°C for 6 hours. A 20 /xL sample was then injected onto the HPLC column and analyzed. (B) Chromatogram obtained from snake venom S -nucleotidase incubated with A(S)MP. In a reaction volume of 100 /xL containing 100 mM Tris-Cl (pH 8.1), 300 jxM A(S)MP, and 20 mM MgCl2, the reaction was initiated by addition of 6 yxg of enzyme and the reaction mixture incubated at 30°C for 60 minutes, and a 20 yxL sample was injected onto the HPLC column and analyzed. (From Rossomando et al., 1983.)...

Figure 10.8 HPLC elution profiles of an incubation mixture to study hypoxanthine/ guanine phosphoribosyltransferase activity. The reaction was initiated by the addition of the enzyme mixture, and aliquots were injected onto the HPLC column at 10-minute intervals. The solid peaks represent hypoxanthine, and the hatched peaks IMP. At 30 minutes, a S -nucleotidase activity was added to the reaction mixture. (From Jahngen and Rossomando, 1984.)...

Immediately after removal of the 30-minute sample, the S -nucleotidase enzyme was added to the incubation tube. The incubation was continued, and samples were again removed for analysis. The HPLC profile in Figure 10.8, obtained from a sample removed after an additional 10 minutes of incubation, shows an increase in inosine, the product of the S -nucleotidase activity, as well as a decline in IMP. Hypoxanthine continues to decline after the reconstitution of the system. 

B-7) (B-7) Pyrimidine S -nucleotidase deficiency. RNA, in this condition, can not be completely degrad in maturing red blood cells. The nucleotides of uridine and cytidine accumulate. This is associated with a hemolytic anemia. In lead poisoning, lead inhibits 5 -nucleotidase and a1.< o mav result in an atieojia. In both... 

Lowenstein, JM, Yu, MK, Naito, Y, Regulation of adenosine metabolism by S -nucleotidase, In Regulatory functions of adenosine, (eds. Berne, RM., Rail, TW and Rubio, R), Martinus Nijhoff Publishers, The Hague, 1983, 117-131. 

Proteins containing glycosylphosphatidylinositol(GPI) anchors (Fig. 1) are widespread in animal cells [2], wliere they are highly localized on the outer face of the plasma membrane. There are over 150 different GPI-anchored proteins known, with alkaline phosphatase, acetylcholinesterase, and S -nucleotidase being noteworthy as extensively studied examples. 

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. 

Kennedy, C, Todorov, LD, Mihaylova-Todorova, S and Sneddon, P (1997) Release of soluble nucleotidases a novel mechanism for neurotransmitter inactivation. Trends Pharmacol. Sci. 18 263-266. 

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). 

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... 

A specific protein inhibitor for 5 -nucleotidase has been purified from E. coli cell cytoplasm (10, 16). It prevents the action of the enzyme on 5 -AMP, ATP, and UDPG. It also inhibits the hydrolysis of 5 -AMP by the 5 -nucleotidases from A. aerogenes, S. sonnei, and S. typhimurium (10). Other Enterobacteriaceae also possess similar intracellular protein inhibitors (18) which inhibit all hydrolytic activities of the 5 -nucleo-tidase of these organisms. The relevance of this inhibitor protein to the action of the enzyme in vivo is not known. 

In 1964, Anraku (2, S) reported the isolation of an enzyme from Escherichia coli B which hydrolyzed ribonudeoside 2, 3 -cyclic phosphates. Enzyme fractions representing a 900-fold purification also possessed 3 -nucleotidase activity. Similar activities have subsequently been purified from Proteus mirabilis (4, 5), halophilic Vibrio algino-lyticus (6, 7), Bacillus subtilis (8), and various Enterobacteriaceae, specifically, Shigella sonnei, Salmonella heidelberg, Serratia marcescens, Proteus vulgaris (9), and others (10). The enzyme from each organism is strikingly similar, but some differences are apparent. 

Although in no case has the enzyme been purified to homogeneity, much evidence exists that the ribonudeoside 2, 3 -cyclic phosphate diesterase activity and the 3 -nucleotidase activity reside in the same protein. Thus, in all cases the ratio of the two activities remained constant throughout purification which has varied from 130-fold for the P. mirabilis enzyme (4) to 2000-fold for the enzyme from V. alginolyticus (6). Anraku (S) found that both activities from E. coli B had the same optimal pH, both showed the same behavior to activators such as Co2+, and to inhibitors [Zn2+, Cu2+, ethylenediaminetetraacetate (EDTA)], both were activated simultaneously by heating at 55° for 5 min and... 

Matsuoka I, Ohkubo S (2004) ATP- and adenosine-mediated signaling in the central nervous system adenosine receptor activation by ATP through rapid and localized generation of adenosine by ecto-nucleotidases. J Pharmacol Sci 94 95-9... 

Schetinger MRC, Morsch VM, Bohrer D (2003) Aluminum Interaction with Nucleotides and Nucleotidases and Analytical Aspects of ist Determination. 104 99-138 Schulz S (2002) Synthesis, Structure and Reactivity of Group 13/15 Compounds Containing the Heavier Elements of Group 15, Sb and Bi 103 117-166 Seifert HJ, Aldinger F (2002) Phase Equilibria in the Si-B-C-N System. 101 1-58 Stalke D, see Mahalakshmi L (2002) 103 85-116... 

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