5 -nucleotidase and

Walters and Loring (88) have purified a 3 -nucleotidase about 50-fold from mung bean sprouts (Phaseolus aureus Roxb.). The enzyme hydrolyzes 3 -AMP, 3 -GMP, 3 -CMP in decreasing order and also hydrolyzes the 3 -phosphate group of coenzyme A. (89), but it has no significant activity for 2 - or 5 -ribonucleotides. For 3 -GMP, 3 -AMP, 3 -UMP, and 3 -CMP, Km values are 0.67, 1.1, 7.7, and 15 mM, respectively. The enzyme preparation also contained acid stable ribonuclease activity (89). Both 3 -nucleotidase and acid ribonuclease were inactivated reversibly at pH 5.0 and by dialysis and this inactivation could be prevented by Zn2+. The two activities were similarly inactivated by heat at pH 5 and 7.5. Such data indicate that the two are metalloproteins— probably zinc metalloproteins. These similarities and other kinetic data provide evidence that the 3 -nucleotidase and ribonuclease activities reside in the same protein. 

The NDP phosphohydrolase acts only on 3, 5 - and 2, 5 -nucleoside diphosphates and specifically removes the 3 -(or 2 -) phosphate. This enzyme also differs from the 3 -nucleotidases and PAPS hydrolases from other sources with respect to the nature of the hydrolase reaction, substrate specificity, metal requirements and inhibitors (Tsang and Schiff, 1976b). 

Phosphodiesterases, phosphatases, 5 -nucleotidases, 3 -nucleotidases and phosphotransferases have been discussed in the section on purine breakdown. Some of these enzymes also act on pyrimidine nucleotides, yielding either the nucleoside or the free base. Cytosine deaminases have been found in yeast and E. coli. Cyti-dine and cytidylic acid deaminases are present in extract of most mammalian tissue. The properties of these enzymes are still poorly understood. 

Nucleotides esterified at the 3 - or 5 -hydroxyl can be identified by the liberation of P by specific 3 -nucleotidase and 5 -nucleotidase, respectively. 

The enzymes found in liver cells (Group I enzymes) include more than a dozen enzymes used in diagnostic laboratories, but those used most commonly are the transaminases (GOT and GPT), which continue to be the most widely used indicators of liver cell integrity. Enzymes found in the biliary cells (Group II) include alkaline phosphatase, glutamyl-transferase, leucine amniopeptidase and 3-nucleotidase. 

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). 

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... 

MYOSIN and MYOSIN ATPase NAD SYNTHETASE 3 -NUCLEOTIDASE 5 -NUCLEOTIDASE... 

The enzyme has been partially purified (70-fold) from 38,000 X 9 supernatant fluid from sheep brain homogenates by Ipata (55-58). Thq enzyme (MW 140,000) is reported to be specific for 5 -AMP and 5 -IMP although the substrate specificity does not appear to have been examined closely. 2 - and 3 -AMP are not hydrolyzed (56). Unlike the enzyme from many sources the brain enzyme does not require divalent cations and indeed Co2+, which stimulates several other 5 -nucleotidases, was inhibitory at 5 mM. The enzyme is strongly inhibited by very low concentrations of ATP, UTP, and CTP (50% inhibition by 0.3 pM ATP) but not by GTP. 2 -AMP, 3 -AMP, and a variety of other nucleoside monophosphates, nucleosides, and sugar phosphates do not inhibit. A kinetic examination of ATP, UTP, and CTP inhibition (56-58) revealed that inhibition curves were sigmoidal, indicating cooperativity between inhibitor molecules and an allosteric type of interaction between inhibitor and protein. The metabolic significance of ATP inhibition is... 

The presence of an enzyme with 5 -nucleotidase activity in extracts of potato were referred to by Heppel (1). The enzyme has been purified 200-fold by Klein (82) and studied kinetically (83). All major 5 -nucleotides are hydrolyzed at similar rates. The preparation also hydrolyzed 3 -nucleotides at a substantial rate (20-30% that of 5 -AMP). However, kinetic data (83) suggested that the purified preparation was perhaps a mixture of specific 5 - and 3 -nucleotidases. 

An enzyme similar to the 3 -nucleotidase of mung bean has been isolated from germinating wheat seedlings and purified 800-fold (90). The preparation possessed DNase, RNase, and 3 -nucleotidase activities. These three activities were similar in pH optima, requirements for Zn2+ and sulfhydryl compounds, stability to storage, temperature inactivation... 

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... 

UMP becomes bound to site B which catalyzes the hydrolysis of the phosphomonoester bond. Adenosine and 3 -AMP by binding at site B could interfere with the breakdown of cyclic 2, 3 -UMP. Similarly, binding of bis (p-nitrophenyl) phosphate at site A could interfere with the breakdown of 3 -AMP. Cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate compete for site A while adenosine competes with 3 -AMP for site B. Unemoto et al. (7) have examined the mutual inhibition of substrates and substrate analogs for the enzyme from halophilic V. alginolyticus. They also concluded that 3 -ribonucleotides and ribonucleo-side 2, 3 -cyclic phosphates are hydrolyzed at different sites. However, because of the nature of the mutual inhibition between 3 -AMP and bis(p-nitrophenyl) phosphate, they suggested that part of the site for the latter substrate overlaps with the 3 -nucleotidase site. At this time the precise mechanism of action of the enzyme is not settled, but clearly there are two active sites, one a 3 -nucleotidase site and a cyclic phosphate diesterase site. Anraku (18) has described this protein as a double-headed enzyme. 

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. 

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