Liver enzymes 5 -nucleotidase

In liver metastases, the serum alkaline phosphatase level shows a better correlation with the extent of liver involvement than those of other liver tests. To differentiate the origin of elevated alkaline phosphatase levels, tests of other liver enzymes may be performed, such as that for 5 -nucleotidase or y-glutamyltransferase. Determination of alkaline phosphatase isoenzymes may provide additional specificity. The liver isoenzyme is thermally more stable than the bone isoenzyme (see Chapter 21 for a more detailed discussion). Other malignancies, such as leuicemia, sarcoma, and lymphoma complicated with hepatic infiltration, may also show elevated ailcaline phosphatase levels. 

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. 

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]. 

Nucleotidases have been studied in liver from various species and activity has been identified in lysosomes, cytoplasmic supernatants and plasma membrane preparations. Arsenis and Touster (31) have purified a 5 -nucleotidase from rat liver lysosomes to apparent homogeneity. The enzyme is unusual in that it hydrolyzes 2 -, 3 -, and 5 -mononucleotides equally well with preference for 5 -dAMP. It also hydrolyzes FMN, p-nitrophenyl phosphate, and /J-glycerol phosphate, but not inorganic pyrophosphate or bis(p-nitrophenyl) phosphate. Unlike the 5 -nucleotidases described thus far, divalent cations such as Co2+, Mn2+, and Mg2+ have no activating effect, but EDTA is inhibitory. In spite of the broad substrate specificity kinetic experiments indicate that a single enzyme is involved. Because of its broad substrate specificity it has been suggested (SI) that it may play a key role in lysosomal catabolism of nucleic acids. 

Thus, there is likely as many as three enzymes with 5 -nucleotidase activity in liver, one lysosomal, one cytoplasmic, and one membrane bound. Their specificities and kinetic properties appear to be distinctly different. This would suggest specialized physiological functions not yet understood. 

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. 

Enzymes in this category include alanine and aspartate aminotransferases, glutamate dehydrogenase (GLD), ATP, 5 -nucleotidase (NTP), y-glutamyl transferase (GGT), glutathione S-transferase (GST), and serum cholinesterase (CHE). The aminotransferases and ALP are widely used. They have long been mistakenly called, as a group, liver function tests. They are not, of course, but the habit persists. GGT is widely available in the United States and on automated analyzers. The others have not been adopted as widely. 

Measurement of serum y-GT activity has clinical significance. The enzyme is present in all tissues, but the highest level is in the kidney however, the serum enzyme originates primarily from the hepatobiliary system. Elevated levels of serum y-GT are found in the following disorders intra- and posthepatic biliary obstruction (elevated serum y-GT indicates cholestasis, as do leucine aminopeptidase, 5 -nucleotidase, and alkaline phosphatase) primary or disseminated neoplasms some pancreatic cancers, especially when associated with hepatobiliary obstruction alcohol-induced liver disease (serum y-GT may be exquisitely sensitive to alcohol-induced liver injury) and some prostatic carcinomas (serum from normal males has 50% higher activity than that of females). Increased activity is also found in patients receiving phenobarbital or phenytoin, possibly due to induction of y-GT in liver cells by these drugs. 

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. ... 

The degradation of the purine nucleotides (AMP and GMP) occurs mainly in the liver (Fig. 41.19). Salvage enzymes are used for most of these reactions. AMP is first deaminated to produce IMP (AMP deaminase). Then IMP and GMP are dephosphorylated (5 -nucleotidase), and the ribose is cleaved from the base by purine nucleoside phosphorylase. Hypoxanthine, the base produced by cleavage of IMP, is converted by xanthine oxidase to xanthine, and guanine is deaminated by... 

Two publications on the higher ketose mono- and bis-phosphates in rat-liver extracts have described their detection and estimation by colorimetry and enzymic assay,and the synthesis of octulose 1,8-bisphosphates using muscle aldolase. A stereospecific synthesis of adenosine 3, 5 -cyclic phosphothioate has appeared/ Standard condensation methods have been used to synthesize the 5 -O-(Taminoethane-phosphonyl) nucleosides (21) and (22). The compounds were shown to be inert to the action of alkaline phosphatase and are poor substrates for 5 -nucleotidase. The selenophosphates (23) and (24) have been prepared.Phosphorylation of nucleosides using dibenzyl hydrogen phosphate,... 

The patient s RBC ADA activity was 43 000 nmol.min . ml RBC " (normal values 495 i 60), There was an about 3-fold increase of red cell pyrimidine 5 -nucleotidase and orotate phosphoribosyl-transferase, whereas other enzymes of purine and pyrimidine metabolism (inosine phosphorylase, adenosine kinase, adenine phosphoribo-syltransferase, hypoxanthine-guanine-phosphoribosyltransferase, phosphoribosylpyrophosphate synthetase) were normal or slightly elevated. There was a 6-fold increase of pyruvate kinase activity relatively to comparably reticulocyte-rich blood, and a 1.5 to 3-fold increase of the other enzymatic activities of glucose and glutathione metabolism. Plasma ADA was much elevated (30.5 pmol.min . ml normal value 5.1 - 2.5), probably reflecting intravascular hemolysis. ADA activity in lymphocytes (2.13 nmol.min 1.10 cells normal 1.93 0.61) and in fibroblasts (26 nmol.min l.mg protein 1 normal range 14-118) was normal, whereas the small increase of activity in platelets (59.5 nmol.min . 10 cells control 26.7) and in the liver (8.4 pmol.min . mg protein" normal ... 

The association of the ethanol effect with the accumulation of a-glycerol-P prompted a study of the influence of this phosphate ester on the enzymes involved in the degradation and the resynthesis of AMP. The activities of liver AMP deaminase, cytoplasmic 5 -nucleotidase and adenosine kinase were not influenced by concentrations of a-glycerol-P up to 10 mM. 

Fig. 4 shows the influence of the conditions prevailing in isolated hepatocytes subjected to anoxia, as compared to the control situation, on the activity of purified rat liver cytoplasmic 5 -nucleotidase. The enhancement of the enzymic activity, resulting from the increase in the concentration of AMP, was completely offset and even reversed, when the concentration of the stimulators was decreased and that of the inhibitor increased, so as to mimic the progress of anoxia. 

Structure of Coemyme A. The elucidation of the structure of CoA depended heavily on d radation by specific enzymes. The phosphate on carbon 3 of the adenosine was shown to be a monoester phosphate by hydrolysis with prostate phosphomonoesterase. The localization of the monoester at the 3 position was established by its sensitivity to a b nucleotidase that attacks only nucleoside 3 -pbosphates, not 2 - or 5 -phosphates. The original CoA molecule or the phosphatase product, depbospho CoA, can be split by nucleotide pyrophosphatases from potato or snake venom. These reactions permitted the identification of the adenosine phosphate portion of the molecule. The position of the phosphate on pantothenic acid cannot be determined enzymatically, but was established by studies on the synthesis of CoA from synthetic phos-phorylated pantetheines. Pantetheine is split to thiolethanolamine and pantothenic acid by an enzyme found in liver and kidney. This enzyme also attacks larger molecules, including CoA. 

Antibodies have been raised against 5 -nucleotidase of mouse liver plasma membranes by using purified membrane preparations from the same source (GURD EVANS, 1974) and the antiserum inhibited enzyme activity. Antisera raised against rat liver as well as rat fat cell plasma membranes inhibited ecto-5 -nucleotidase in isolated rat fat cells (NEWBY al., 1975). However, it is not known whether the immune-specific and the catalytic site of the enzyme are identical. 

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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