5 -Nucleotidase intestine

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... 

Center and Behai (49) have resolved 5 -nucleotidase from calf intestinal mucosa into three fractions using DEAE-cellulose chromatography. One of these was obtained free of nonspecific phosphatase. It had a pH optimum of 6-6.5, Mn2+, Mg2+, and Co2+ (1-10 mill) all enhanced activity and complete inactivation was produced with 1 mM EDTA. This enzyme hydrolyzes all 5 -ribonucleotides at similar rates and hydrolyzes 5 -deoxribonucleotides more slowly. These properties indicate that it is strikingly similar to the one obtained from acetone powder preparations of chicken and rat liver (32, 33) and from soluble supernatants of rat liver (36). The other two activities (which were not fully characterized) (49) could possibly have originated from particulate material or membranes because the authors employed deoxycholate in the early phase of purification. 

Nucleotidase present in 48,000 X Q supernatant fractions of rat and guinea pig skeletal muscle extracts has been examined briefly (7-4). 5 -UMP seems to be the preferred substrate. The enzyme from fish skeletal muscle has also been studied (75). This enzyme hydrolyzes all ribo-and deoxyribonucleoside 5 -phosphates (except dCMP and dTMP) with preference for 5 -IMP and 5 -UMP. The enzyme is strongly activated by Mn2+ Mg2+ is a less powerful activator, and Zn2+ and EDTA are inhibitors. This enzyme thus appears similar to the soluble activity from mammalian liver (88, 86). 5 -Nucleotidase in mammary gland hydrolyzes all 5 -ribonucleotides and shows a decrease from pregnancy to early lactation (76). Rats injected with glucagon show increased 5 -nucleotidase in pancreatic islet tissue (77). The enzyme in mouse kidney has been examined histochemically and electrophoretically and found to exist as isozymes (75). Electrophoretic techniques have also provided evidence that the enzyme exists as isozymes in many other tissues of the mouse such as liver, spleen, intestine, testes, and heart (79). 

It appears certain that there is more than one 5 -nucleotidase present in most mammalian tissues. This is best established for liver. In other cases it has not been possible to determine the exact intracellular origin because of the nonselective extraction procedures used. However, those enzymes isolated from acetone powder preparations of chicken liver and rat liver appear to have properties essentially identical to the enzyme present in 100,000 X ff supernatant fraction of rat liver and therefore may be cytoplasmic in origin. This could also be the case for the intestinal mucosa enzyme. 

In the small intestine, ribonuclease and deoxyribonuclease I, which are secreted in the pancreatic juice, hydrolyze nucleic acids mainly to oligonucleotides. The oligonucleotides are further hydrolyzed by phosphodiesterases, also secreted by the pancreas, to yield 5 - and 3 -mononucleotides. Most of the mononucleotides are then hydrolyzed to nucleosides by various group-specific nucleotidases or by a variety of nonspecific phosphatases. The resulting nucleosides may be absorbed intact by the intestinal mucosa, or they may un-... 

Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Eltzschig HK, Hansen KR, Thompson LF, Colgan SP (2002) Ecto-5 -nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest 110(7) 993-1002... 

Most of the enzymes involved in the hydrolysis of dietary DNA and RNA are secreted in the intestine. Thus, ribonucleases are found in the lumen of the small intestine, while the phosphodiesterases and nucleotidases are present in the mucosal cells. 

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.)...

Levene and Dillon discovered that the gastro-intestinal secretions of the dog contain a polynucleotidase and a non-specific nucleotidase. Whereas uridylic acid is the most stable of the natural ribosenucleotides as regards hydrolysis with mineral acids, it is more readily dephosphory-lated by nucleotidase than is any other nucleotide. 

In 1933, Klein discovered that sodium arsenate inhibits the action of the nucleotidase" of intestinal mucosa. Consequently, Klein and Thannhauser were enabled to hydrolyze thymus nucleic acid by means of intestinal desoxyribonucleinase without subsequent dephosphorylation of the liberated nucleotides. Making use, also, of Klein s discovery of the deaminase-inhibiting activity of silver ions, they were successful in isolating the adenine nucleotide. Hence the phosphodesoxyribosyl nucleotides of adenine, guanine, thymine, and cytosine were isolated and characterized. 

Previous work on human alkaline phosphatases has utilized chromatography (ElO) and starch-gel electrophoresis. Thus in 1956 Boman and Westlund (B34) reported the purification and separation of serum phosphatases by Dowex-2 column chromatography. Moss (M34) used gel filtration on Sephadex G-200 and DEAE-celluIose chromatography for separating 5 -nucleotidase and nonspecific alkaline phosphatase activities in human sera. In most of the studies of alkaline phosphatases in human tissues of liver (M33), intestine (M34, M35), bone (M36), kidney (B46), and urine (B44, B46, B47), crude extracts of these tissues were used and... 

Often there is no good clinical test available to determine the exact type of hepatic lesion, short of liver biopsy. There are certain patterns of enzyme elevation that have been identified and can be helpful (Table 38-3). ° The specificity of any serum enzyme depends on the distribution of that enzyme in the body. Alkaline phosphatase is found in the bile duct epithelium, bone, and intestinal and kidney cells. 5-Nucleotidase is more specific for hepatic disease than alkaline phosphatase, because most of the body s store of 5 -nucleotidase is in the liver. Glutamate dehydrogenase is a good indicator of centrolobular necrosis because it is found primarily in centrolobular mitochondria. Most hepatic cells have extremely high concentrations of transaminases. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are commonly measured. Because of their high concentrations and easy liberation from the hepato-cyte cytoplasm, AST and ALT are very sensitive indicators of necrotic lesions within the liver. After an acute hepatic lesion is established, it may take weeks for these concentrations to return to normal. ... 

During digestive processes, nucleoprotein is split into nucleic acids and protein, the latter then being broken down into amino acids. The nucleic acids are attacked by ribonuclease and deoxyribonuclease enzymes to form nucleotides, which are further hydrolysed by nucleotidases to form nucleosides and phosphates. In the intestines these nucleosides are split by nucleosidases into ribose, deoxy-ribose, purine and pyrimidine bases, which later undergo oxidation and decomposition to ammonia, carbon dioxide and water, to be finally expelled as urea. Nucleotide hydrolysis products are conveniently identified and isolated by chromatographic methods (Chapter 14.2). 

The phosphate group of a purine nucleotide can be lost in at least three different types of reactions catalyzed by phosphatases with broad specificity, nucleotidases, and transferases. Intestinal and liver phosphatase, prostatic acid phosphatase, and bone phos-phomonoesterase hydrolyze purine 5 - and 3 -mononucleotides. A 5 -nucleotidase acting on 5 -AMP, 5 -UMP nicotinamide 5-nucleotide and R5P prepared from bull has been studied more extensively. It acts optimally at pH 8.5 and is activated by magnesium and inhibited by fluorides and borates. 

Nucleotidases are phosphatases active against nucleotides, and such activity is widely distributed in nature. Among mammalian tissues one may mention alkaline intestinal phosphatase,acid prostatic phosphatase, and alkaline bone phosphatase. None of these preparations are specific for nucleotides, although extracts of prostate dephosphorylate nucleotides at a much faster rate than other esters. Enzymes which act only on nucleotides may occur in these tissues but their existence has not been proved. Several specific nucleotidases will now be discussed. 

Dietary nucleic acids first pass through the stomach to the intestines, where they are hydrolyzed to their constituent nucleotides by a variety of different nucleases. Depbosphorylation by various nucleotidases next gives nucleosides, and cleavage by nucleosidases then gives the constituent bases, which are catabolized to produce intermediates that enter other metabolic processes or are excreted. 

Intestinal juice. Alkaline pH 9. Erepsin. Sucrase. Lactase. Nucleinase. Nucleotidase. Enterokinase. Peptides. Sucrose. Lactose. Nucleic acids. Nucleotides. Irypsinogen. Amino acids. Glucose + fructose. Glucose + galactose. Nucleotide. Nucleoside -1- H.PO,. Trypsin. 

The following three characteristic mammalian liver and kidney enzymes are absent from muscle catalase, xanthine oxidase, and D-amino oxidase. The distribution of many other enzymes in mammals is limited to particular organs. Thus arginase occurs only in the liver, alkaline phosphatase in the intestinal mucosa, acid phosphatase in kidney, spleen, and prostate, 5-nucleotidase in the testis, and a-mannosidase in the epididymis (see Table 4.6). The blood is disproportionately rich in carbonic anhydrase, and the pancreas in ribonuclease. Glutamine synthetase, which condenses... 

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