Enzyme 5 -nucleotidase

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],... 

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... 

Steady state concentrations of adenosine are maintained through the activities of only three enzymes, 5 -nucleotidase (5 -N), adenosine kinase and adenosine deaminase. Adenosine kinase and adenosine deaminase were located mainly in the soluble fractions of rat cerebellar homogenates, whereas 5 -N was present in subcellular fractions (Philips and Newsholme, 1979), mainly in the synaptosomal fraction (Marani, 1977). Adenosine deaminase-immunoreactivity in rat cerebellum was present with one out of five polyclonal sera prepared by Nagy et ah (1988). Staining was present in most Purkinje cells with a variation in intensity. Staining was observed in the Purkinje cell axons and terminals in the cerebellar and vestibular nuclei. The localization of 5 -N will be discussed below. 

The distribution of the Zebrin-positive Purkinje cells was very similar to the distribution of the enzyme 5 -nucleotidase in the molecular layer of certain rodents (Eisenman and Hawkes, 1989). 

Inhibition of EGF-R tyrosine kinase by hypericin 1 was shown to be irreversible, non-competitive and time as well as temperature dependent. The IC50 increased from 0.75 pM in the dark to 44 nM with light illumination for 30 min. This effect was presumably due to a type I photosensitization mechanism since exclusion of oxygen did not alter the inhibition curve. Some Ser/Thr protein kinases (e.g., protein kinase A, casein kinase 1 and 2) and the enzyme 5 -nucleotidase were not inhibited even at concentrations > 100 pM [144]. However, the same authors recently reported that hypericin 1 in addition to protein kinase C also caused the light-dependent inhibition of certain other Ser/Thr kinases (e.g. protein kinase CK-2, mitogen-activated kinase) and the insulin receptor tyrosine kinase, while it was ineffective towards the cytosolic tyrosine kinases Lyn, Fgr, TPK-IIB and CSK. These results suggest that distantly related protein kinases could still share common reactive domains for the interaction with hypericin 1 [156]. In contrast to the above mentioned studies, Richter and Davies [157] observed no inhibition of EGF-induced tyrosine phosphorylation of the EGF-R in HN5 squamous carcinoma... 

Enzymes that cleave phosphate from certain organic molecules may not be produced principally to satisfy a phosphorus limitation (Bjorkman and Karl, 1994). For example, the enzyme 5 -nucleotidase recognizes the C-N group of DNA and RNA, yet it has been shown to release significant amounts of phosphate (Benitez-Nelson and Buesseler, 1999). Benitez-Nelson and Bues-seler (1999) found rapid rates of turnover for filterable reactive phosphorus and filterable... 

Although several enzymes can in theory convert purine ribonucleotides to ribonucleosides, in only a few cases is it known exactly which is acting. Certain strains of Bacillus svbtilis accumulate nucleotides extra-cellularly, but mutants lacking 5 -nucleotidase or alkaline phosphatase, or both, accumulate smaller quantities of nucleotides than cells which have both enzymes. 5 -Nucleotidase appeared to be quantitatively the more important for nucleotide dephosphorylation in these cells 23). Baer and Drummond 12) compared the rates of dephosphorylation of the 2 -, 3 -, and 5 -phosphates of adenosine by perfused rat heart and concluded that only a specific 5 -nucleotidase was actively in contact with the blood. More evidence for the action of specific, rather than nonspecific phosphatases, are the observations that in one system or another, adenylate, inosinate, xanthylate, or guanylate appears to be the nucleotide most rapidly de-phosphorylated. 

Gibson, S.L., Murant, R.S., and Hilf, R., Photosensitizing effects of hematoporphyrin derivative and photofrin II on the plasma membrane enzymes 5 -nucleotidase, Na+K+-ATPase, and Mg -ATPase in R3230AC mammary adenocarcinomas. Cancer Res., 48, 3360, 1988. 

The ATPase of most biological systems hydrolyzes the a-phosphate producing ADP + Pj. When ATP is used as a substrate, snake venom exonuclease hydrolyzes it to AMP and PP (pyrophosphate). However, when ATP is mixed with snake venom, it is hydrolyzed to adenosine, Pj and PP because snake venom contains an additional enzyme, 5 -nucleotidase. This can be summarized as follows ... 

The drug is a pyrimidine nucleoside antimetabolite which is cytotoxic to a plethora of cell-types. Precisely the induction of the enzyme nucleotidase into DNA inhibits polymerization via termination of strand synthesis. It is S-phase specific. 

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 ... 

The RBC contains certain enzymes of nucleotide metabolism (eg, adenosine deaminase, pyrimidine nucleotidase, and adenylyl kinase) deficiencies of these enzymes are involved in some cases of hemolytic anemia. 

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. 

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). 

P5N has two isozymes, P5N-I (pyrimidine nucleotidase) and P5N-II (deoxyri-bonucleotidase) (H6, P2). P5N-I is active principally with pyrimidine substrates at an optimal neutral pH P5N-II activity occurs with both purine and pyrimidine substrates and was maximal with deoxy analogues at an acidic pH optimum. This enzyme was partially purified from human red blood cells and had a molecular weight of 28,000 (T19). The primary structures of both isozymes have not been... 

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 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... 

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. 

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... 

Using phosphotriester methods, dinucleoside (3 - 50-monophosphates containing 6-methyl-2,-deoxyuridine at the 3 - or 5 -end have been prepared.44 N.m.r. spectroscopy indicates that this nucleoside possesses the syn conformation in these compounds, and, on treatment with snake venom phosphodiesterase, d(m6UpT) is degraded, while d(Apm6U) is not, indicating that this enzyme, a 3 -exonuclease, requires the anti conformation to be present in the substrate. Two modified nucleo-side-5 -monophosphates, (20) and (21), which are resistant to 5 -nucleotidase, have been isolated from tRNA snake venom hydrolysates.45 A synthesis of (20) has been reported.46... 

Both the 26S proteasome and the RC hydrolyze all four nucleotide triphosphates, with ATP and CTP preferred over GTP and UTP [58]. Although ATP hydrolysis is required for conjugate degradation, the two processes are not strictly coupled. Complete inhibition of the peptidase activity of the 26S proteasome by calpain inhibitor I has little effect on the ATPase activity of the enzyme. The nucleotidase activities of the RC and the 26S proteasome closely resemble those of E. coli Lon protease, which is composed of identical subunits that possess both proteolytic and nucleotidase activities in the same polypeptide chain. Like the regulatory complex and 26S proteasome, Lon hydrolyzes all four ribonucleotide triphosphates, but not ADP or AMP [18]. 

In addition to the enzymes that catalyse the formation of nucleotides and polynucleotides, a large number of catabolic systems exist which operate at all levels of the internucleotide pathways. The ribonucleases and deoxyribonucleases that degrade polynucleotides are probably not significantly involved in purine analogue metabolism, but the enzymes which dephosphorylate nucleoside 5 -monophosphates are known to attack analogue nucleotides and may be of some importance to their in vivo activity. Phosphatases of low specificity are abundant in many tissues [38], particularly the intestine [29]. Purified mammalian 5-nucleotidases hydrolyse only the nucleoside 5 monophosphates [28] and... 

Ford JH, Evans J. 1985. Distribution of 5 -nucleotidase in the tissues of sheep and the effect of kidney and liver lesions on the activity of enzyme in plasma and urine. Res Vet Sci 39 103-109. 

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]. 

Biochemical studies have also suggested an asymmetric orientation of constituents in lipid globule membrane. By comparison of specific activities of enzymes in washed lipid globules and released membrane, Patton and Trams (1971) suggested that the active site of Mg2+-adeno-sine triphosphatase was accessible to substrates on both faces of the membrane and that of 5 -nucleotidase on the outer membrane face. Recent evidence from studies of Concanavalin A inhibition of globule membrane and plasma membrane 5 -nucleotidase support an outer surface localization for the active site of this enzyme (Carraway and Carra-way 1976 Snow et al. 1980). Kobylka and Carraway (1973) observed that exposure of lipid globules to proteolytic enzymes resulted in cleavage of all major membrane-associated proteins. They concluded that... 

First, exonuclease (phosphodiesterase), endonuclease (8, 10-12), 5 -nucleotidase (13), and nonspecific phosphatase (13) are present in all venoms that have been analyzed. Therefore, nature did not provide these materials as sources of any one of these enzymes free from undesirable contaminants which may be represented by the others. 

One of the monophosphatases, 5 -nucleotidase, is quite specific and attacks only 5 -mononu cleotides. This enzyme has been used to determine the amount of mononucleotides in a mixture of 5 -monophosphates of various chain length. It was also used to distinguish between 3 - and 5 -mononucleotides in a mixture of both. 

Nucleotidase (5 -ribonucleotide phosphohydrolase, EC 3.1.3.5) is widely distributed in nature and a voluminous literature has appeared in the past decade on the enzyme from vertebrate tissues, seminal fluid, snake venoms, yeasts, and bacteria. Studies regarding the discovery and early investigations of the enzyme have been reviewed by Heppel (1) and... 

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. 

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