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PubMed1987

Properties of the membrane-bound 5'-nucleotidase and utilization of extracellular ATP in Vibrio parahaemolyticus.

Vibrio parahaemolyticus utilized ATP, ADP or AMP as the sole source of carbon. About three times higher activity of membrane-bound 5'-nucleotidase was observed in cells grown in the presence of these nucleotides than in their absence: and therefore the enzyme seems to be inducible. Since the 5'-nucleotidase activity could be measured with whole cells, the active site of this enzyme appears to be outwardly oriented. Both Mg2+ and Cl- were required for activity. Among the divalent cations tested, Mn2+ and Co2+ could replace Mg2+ to some extent, whereas Zn2+ strongly inhibited activity. Among the anions tested, Br-, I- and NO3- could replace Cl-, but SO4(2-) and CH3COO- could not. When cells were grown with ATP, Cl- was indispensable and Zn2+ strongly inhibited growth. Therefore, it is concluded that extracellular ATP and other 5'-nucleotides are cleaved by the membrane-bound 5'-nucleotidase outside the cells and that the adenosine produced is then utilized.

5'-Nucleotidase↗
PubMed1980

Erythrocyte pyrimidine 5'-nucleotidase.

In this study 31 family members of a patient with erythrocyte pyrimidine 5'-nucleotidase deficiency were studied. The activity of this enzyme in their erythrocytes is compared with levels in normal subjects and the problems surrounding heterozygote detection are discussed. The mean erythrocyte pyrimidine 5'-nucleotidase activity in 158 normal fresh blood samples was 130 +/- SD 29.8 mU/gHb. There was no significant difference between males and females. The enzyme level in a patient with non-spherocytic haemolytic anaemia was 6 mU/gHb. Of 31 relatives of the enzyme deficient patient examined five were clearly heterozygous for the enzyme defect. Their enzyme levels were below 50 mU/gHb. The father, who is an obligatory heterozygote, had an enzyme level of 87 mU/gHb which falls within the low values of the normal range. The distribution of enzyme activity in 26 family members having enzyme activities greater than 50 mU/gHb suggests that six of these may be carriers of the defective gene. We were unable to identify carriers by enzyme kinetic studies, electrophoresis, chromatographic examination of acid extractable nucleotides or measurement of enzyme levels in young and old erythrocyte populations. The last-mentioned technique showed that erythrocyte 5'-nucleotidase activity in reticulocytes may be as high as 1785 mU/gHb and declines rapidly as the cell ages reaching about 50 mU/gHb in the oldest cells. Blood samples which had been stored frozen were examined to see whether such samples were satisfactory for population studies. The mean enzyme activity 151 blood samples stored frozen for more than 12 months was 153+/-SD 44.7 mU/gHb. The increase in enzyme levels in the frozen samples appears to be greatest in samples showing haemolysis. In spite of the increased enzyme level frozen samples could be used to detect subjects with enzyme deficiency and some heterozygotes.

5'-Nucleotidase↗
PubMed1983

Histochemical and biochemical study on adenylate cyclase and 5'-nucleotidase activity in thyroid glands with normal and various thyroid diseases.

The adenylate cyclase and 5'-nucleotidase activity was measured biochemically in the thyroid glands from patients with various thyroid diseases in comparison with normal thyroid. The basal adenylate cyclase activity in normal thyroid was 159.3 p-moles cAMP/min./g tissue. The activity was elevated to 230% of basal with 20 mM NaF and 190% of basal with 100 mU/ml TSH. These values in chronic thyroiditis and Graves' disease were not significantly different from the values of normal thyroid. In adenomatous goiter, adenoma and carcinoma, the basal adenylate cyclase activity was significantly higher than that of normal thyroid. Parallel to the biochemical determination of both enzyme activities, the distribution of histochemically demonstrable adenylate cyclase and 5'-nucleotidase activity was described in the follicular cells with normal and various thyroid diseases. The reaction product of adenylate cyclase and 5'-nucleotidase activity was restricted to the plasma membrane of the follicular cells. However, the distribution and intensity of the adenylate cyclase reaction varied in each thyroid disease, except for the absence of reaction product in the basal plasma membrane. The lack of demonstrable adenylate cyclase activity in the basal plasma membrane suggests the possibility that the basal plasma membrane may not play an important role of TSH-reception.

5'-Nucleotidase↗
PubMed1983

Presynaptic distribution of the cholinergic-specific antigen Chol-1 and 5'-nucleotidase in rat brain, as determined by complement-mediated release of neurotransmitters.

Nerve terminals prepared from rat cortex and hippocampus were loaded with seven radioactive putative neurotransmitters (serotonin, noradrenaline, dopamine, gamma-aminobutyric acid, aspartate, glutamate, and taurine). The release of these transmitters, choline acetyltransferase, 3,4-dihydroxyphenylalanine decarboxylase, enolase, and lactate dehydrogenase was monitored during complement-mediated lysis. Three antisera were used: anti-5'-nucleotidase, anti-Chol-1, and anti-rat cerebrum. Anti-5'-nucleotidase serum did not cause the release of any labelled transmitter or of any of the enzymes studied. Anti-Chol-1 serum released choline acetyltransferase and small amounts of enolase and lactate dehydrogenase. Anti-rat cerebrum caused the release of all seven transmitters, choline acetyltransferase, and small amounts of the other three enzymes. It was concluded that 5'-nucleotidase was not present on any of the terminals studied, and that Chol-1 is only present on cholinergic terminals.

5'-Nucleotidase↗
PubMed1984

5'-Nucleotidase from bovine caudate nucleus synaptic plasma membranes: specificity for substrates and cations; study of the carbohydrate moiety by glycosidases.

We studied 5'-nucleotidase in preparations of synaptic plasma membranes from bovine caudate nucleus. The best substrates for this membrane-bound enzyme were purine nucleotides, particularly 5'AMP. Effects of metal cations and chelating agents suggest that 5'-nucleotidase is a metalloprotein. Optimal conditions for solubilization of the 5'-nucleotidase were found by using a low concentration of the zwitterionic detergent sulfobetaine 14. In contrast, another membrane-bound enzyme, acetylcholinesterase, was not solubilized under these conditions, but only in the presence of Triton X-100. The effects of lectins (concanavalin A, Lens culinaris agglutinin, wheat germ agglutinin, and Limulus polyphemus agglutinin) showed that both enzymes are glycoproteins. Sequential hydrolysis with specific glycosidases produced modifications of the effect of lectins on these enzymes. The results suggest the presence of a complex-type glycosylation, with a fucose residue on the internal N-acetyl-D-glucosamine of the pentasaccharide core.

5'-Nucleotidase↗
PubMedDate not supplied

Comparison of 5'-nucleotidase activities among cultured murine melanoma cell lines.

The activity of 5'-nucleotidase and ouabain-sensitive Na/K ATPase was determined in seven different mouse melanoma cell lines. Ouabain-sensitive Na/K ATPase activity was found in NP40-treated cell homogenates of all cell lines. However, 5'-nucleotidase activity was found in only one mouse melanoma cell line--JB/RH. The absence of expression of 5'-nucleotidase activity in the other six cell lines is not associated with pigmentation in melanoma cells, nor is the gene switched off in all transformed melanocytes of C57BL/6 origin.

5'-Nucleotidase↗
PubMed1984

Kinetics of pulmonary angiotensin-converting enzyme and 5'-nucleotidase in vivo.

Angiotensin-converting enzyme and 5'-nucleotidase line the luminal surface of pulmonary microvascular endothelium and participate in the synthesis and/or degradation of potent vasoactive substances. We applied Michaelis-Menten kinetics in simultaneous estimations of apparent constants Km and Amax (product of Vmax and microvascular plasma volume) of these two enzymes for the substrates 3H-labeled benzoyl-Phe-Ala-Pro and 14C-labeled 5'-AMP, respectively, in vivo. Values of angiotensin-converting enzyme for benzoyl-Phe-Ala-Pro (Km = 10-11 microM; Amax = 12-13 mumol X min-1) were somewhat higher than published estimates in vitro and changed predictably in response to the known enzyme inhibitor captopril. Kinetic values of 5'-nucleotidase for 5'-AMP (Km = 3-4 microM; Amax = 3-4 mumol/min) were substantially lower than those reported in vitro but also responded predictably to the competitive inhibitor of 5'-nucleotidase, adenosine 5'-[alpha, beta-methylene]diphosphate. These data offer in vivo estimates of enzyme kinetics that are useful in revealing enzyme behavior in their normal physiological environment and provide means of evaluating the action of pharmacological, physiological, and pathological modulators of enzyme activity, in vivo.

5'-Nucleotidase↗
PubMed1985

Properties of an ecto-5'-nucleotidase of the renal brush border.

A decrease of glomerular filtration rate can be observed during accelerated catabolism of ATP in kidney. It has been proposed that this effect is due to the increase in the renal production of adenosine from ATP. The last reaction in the pathway concerned is the conversion of 5'-AMP to adenosine. We found that brush border membranes purified from homogenates of the rat renal cortex carry out this reaction. The enzyme involved in the hydrolysis has the characteristic properties of ecto-5'-nucleotidases: It is inhibited by ATP, ADP, and by alpha, beta-methyleneadenosine-5'-diphosphate, and it is not stimulated by magnesium. All catalytic sites are accessible from the outside of the vesicles. The Km of the enzyme for 5'-AMP is 5.77 microM. The enrichment of the 5'-AMP-hydrolyzing activity in the brush border fraction as compared to the homogenate is 9.2 +/- 1.5 times. Histochemical staining of kidney sections reveals hydrolysis of 5'-AMP only at the brush border of the proximal tubule. We conclude that the brush border of the proximal tubule of the rat kidney possesses an ecto-5'-nucleotidase which has the same properties as the ecto-5'-nucleotidases in other tissues.

5'-Nucleotidase↗
PubMed1983

Ultrastructural localization of 5'-nucleotidase in guinea pig neutrophils based upon the use of cerium as capturing agent.

A cytochemical method for 5'-nucleotidase localization in which cerium serves as the capture agent in order to enzymatically detect liberated inorganic phosphate has been developed. The method has been established in cell-free model systems and in guinea pig neutrophils where 5'-nucleotidase is restricted to the plasmalemma as an ectoenzyme. This cerium-based method gives better results for ultrastructural localization of 5'-nucleotidase than conventional lead-based methods.

5'-Nucleotidase↗
PubMed1988

Immunocytochemical localization of cytosol 5'-nucleotidase in chicken liver.

We investigated the localization of cytosol 5'-nucleotidase in chicken liver by use of a pre-embedding immunoenzyme technique. Cytosol 5'-nucleotidase was purified from chicken liver and a monospecific antibody to this enzyme was raised in a rabbit. Fab fragments of the antibody were conjugated with horseradish peroxidase. Tissue sections of the fixed chicken liver were incubated with the peroxidase-Fab fragments, followed by DAB reaction for peroxidase. By light microscopy, dark-brown staining was present in the cytoplasm of parenchymal cells, Kupffer cells, and endothelial cells. The latter two types of cells were stained more strongly than the former. By electron microscopy, reaction deposits were present in the cytoplasmic matrix but not in cell organelles, such as mitochondria, endoplasmic reticulum, and peroxisomes, or in nuclei. In control sections incubated with peroxidase-conjugated Fab fragments from non-immunized rabbit, no specific reaction was noted. The results indicate that cytosol 5'-nucleotidase is contained more in the sinus-lining cells and less in the parenchymal cells, and that the enzyme is present in the cytoplasmic matrix of these cells.

5'-Nucleotidase↗
PubMed1980

Cell surface localization of 5'AMP nucleotidase in prestalk cells of Dictyostelium discoideum.

5'AMP nucleotidase activity was localized by electron microscopy in Dictyostelium discoideum during cell differentiation. In addition, the activity was assayed by micro enzymic methods in sections dissected from specific cellular regions of lyophilized individuals. The results of the 2 procedures were in agreement, demonstrating that at the culmination stage of development the activity is strikingly localized in the prestalk cells adjacent to the prespore region. The cytochemically stained reaction product appeared only along the plasma membrane of the cells. As prestalk cells migrate into the stalk sheath and undergo differentiation, the activity is rapidly lost. Examination of stained cells at high magnification revealed the product accumulation to be primarily at the cell surface, suggesting that the enzyme functions extracellularly. Occasionally, cells having the morphological appearance of prestalk cells were found within the prespore region. These cells demonstrated 5'AMP nucleotidase activity at their plasma membrane in sharp contrast with neighbouring prespore cells. The strategic localization of 5'AMP nucleotidase may reflect a mechanism for establishing and maintaining regulatory levels of extracellular 5'AMP and/or adenosine during pattern formation in this model system.

5'-Nucleotidase↗
PubMed1981

Histochemical study on 5'-nucleotidase in the cerebrovascular system in stroke-prone spontaneously hypertensive rats.

5'-Nucleotidase activity in the cerebrovascular system in stroke-prone spontaneously hypertensive rats was histochemically investigated. In the hypertensive rats, an enhancement of 5'-nucleotidase activity was observed already in the early stage of hypertension and the enzyme activity increased with advancing age. The enzyme activity appeared earlier and stronger in the larger arteries in the basal portion than the pial arteries in the convexity of the brain and arterioles in the brain parenchyma. The activity in the arterial walls proved to be particularly strong in the thickened parts showing cellular hyperplasia, mainly at the branching portions. The reaction products localized along the plasma membrane, and also in the cytoplasma in some parts where the activity was strong. Relationship between the increased 5'-nucleotidase activity and aging of the cells composing the cerebrovasculature resulted from an accelerated or repeated cell proliferation was discussed.

5'-Nucleotidase↗
PubMed1989

Human placental ecto-5'-nucleotidase: isoforms and chemical crosslinking products of the membrane-bound and isolated enzyme.

Human placental ecto-5'-nucleotidase is specifically detected on Western blots using poly- or monoclonal antibodies. In two-dimensional electrophoresis 5'-nucleotidase, purified as well as membrane bound, is resolved in up to 13 isoforms distinguished by a different content of neuraminic acid. These forms span a range of molecular masses of about 67-71 kDa and of isoelectric points of 5.8-7.0. Chemical cross-linking of the purified enzyme with either homoor heterobifunctional reagents yields a dimer of about 140 kDa exclusively. On the other hand treatment of plasma membranes with the same reagents results in a crosslinking product of 5'-nucleotidase of about 97 kDa. The partner of the enzyme subunit in this crosslink, a protein of about 30 kDa, is unknown. Using specific antibodies the cytoskeletal components actin and tropomyosin were excluded as possible candidates.

5'-Nucleotidase↗
PubMedDate not supplied

Conformation about the glycosidic bond and susceptibility to 5'-nucleotidase of 8-substituted analogues of 5'-GMP.

Susceptibilities to snake venom 5'-nucleotidase (EC 3.1.3.5) have been evaluated for several 8-substituted analogues of 5'-GMP with varying populations of syn/anti conformations about the glycosidic bond. Improved syntheses of some of these are described, including direct chlorination of 5'-GMP to give 8-chloro-5'-GMP, a procedure which should be applicable to other purine nucleotides. The conformations of the various analogues were determined by means of 1H NMR spectroscopy, with particular emphasis on the glycosidic bond conformations. All the 8-substituted derivatives of 5'-GMP were relatively poor substrates of 5'-nucleotidase. This was shown to result largely from steric effects and the nature of the 8-substituent, and consistent with a requirement for the anti conformation. Although ribose-5-phosphate was not a substrate, it was a weak inhibitor, and its inhibitory properties account in part for the weak inhibitory properties of the 8-substituted 5'-GMP, and other, analogues. Attention is drawn to the hitherto largely neglected differences in properties of 5'-nucleotidases from different sources and their relevance to the present findings.

5'-Nucleotidase↗
PubMed2006

Ecto-5'-nucleotidase (CD73)-mediated adenosine production is tissue protective in a model of bleomycin-induced lung injury.

Adenosine signaling has diverse actions on inflammation and tissue injury. Levels of adenosine are rapidly elevated in response to tissue injury; however, the mechanisms responsible for adenosine production in response to injury are not well understood. In this study, we found that adenosine levels are elevated in the lungs of mice injured by the drug bleomycin. In addition, increased activity of ecto-5'-nucleotidase (CD73) was found in the lungs in conjunction with adenosine elevations. To determine the contribution of CD73 to the generation of adenosine in the lung, CD73(-/-) mice were subjected to bleomycin challenges. Results demonstrated that CD73(-/-) mice challenged with bleomycin no longer accumulated adenosine in their lungs, suggesting that the primary means of adenosine production following bleomycin injury resulted from the release and subsequent dephosphorylation of adenine nucleotides. CD73(-/-) mice challenged with bleomycin exhibited enhanced pulmonary inflammation and fibrosis as well as exaggerated expression of proinflammatory and profibrotic mediators in the lung. Intranasal instillations of exogenous nucleotidase restored the ability of lungs of CD73(-/-) mice to accumulate adenosine following bleomycin challenge. Furthermore, these treatments were associated with a decrease in pulmonary inflammation and fibrosis. CD73(+/+) animals challenged with bleomycin and supplemented with exogenous nucleotidase also exhibited reduced inflammation. Together, these findings suggest that CD73-dependent adenosine production contributes to anti-inflammatory pathways in bleomycin-induced lung injury.

5'-Nucleotidase↗
PubMed1963

SERUM LACTIC DEHYDROGENASE, LEUCINE AMINOPEPTIDASE AND 5-NUCLEOTIDASE ACTIVITIES: OBSERVATION IN PATIENTS WITH CARCINOMA OF THE PANCREAS AND HEPATOBILIARY DISEASE.

Serum lactic dehydrogenase, leucine aminopeptidase, 5-nucleotidase and alkaline phosphatase activities were investigated in a number of diseases involving the hepatobiliary system.Leucine aminopeptidase was found to be a sensitive indicator of biliary obstruction, serum 5-nucleotidase slightly less sensitive, and alkaline phosphatase appreciably less sensitive. Leucine aminopeptidase and 5-nucleotidase activities were often increased by malignant infiltration of the liver and primary hepatic disease even in the absence of jaundice.Serum lactic dehydrogenase was frequently increased in primary hepatic disease and malignant disorders but was not apparently affected by bile duct obstruction per se. Thirty-five of 45 patients with proved malignancy had increased lactic dehydrogenase levels.The highest leucine aminopeptidase levels were encountered in carcinoma of the head of the pancreas. The frequent increase in both serum lactic dehydrogenase and leucine aminopeptidase activities in patients with carcinoma of the head of the pancreas suggests that these combined estimations are useful laboratory procedures in the diagnosis of malignant extrahepatic obstruction.

Aminopeptidases↗
PubMed1987

Substrate specificity and pH sensitivity of deoxyribonucleotidase and pyrimidine nucleotidase activities in human hemolysates.

Residual 5'-nucleotidase activities in hemolysates from nine subjects with severe hereditary deficiency of pyrimidine nucleotidase (PyrNase) were compared to those in normal and reticulocyte-rich controls. Dephosphorylation rates of 12 potential ribo- and deoxyribomononucleotide substrates were measured as a function of pH. Data confirmed the existence of at least two isozymes of 5'-nucleotidase, PyrNase, and 2'-deoxy-5'-ribonucleotide phosphohydrolase (dNase) distinguishable by differences in maximal velocities, substrate preferences and restrictions, and pH optima. PyrNase was confirmed to be active principally with pyrimidine substrates (UMP = dCMP greater than CMP much greater than dTMP greater than dUMP) at a pH optimum of 7.5 +/- 0.1. dNase activity occurred with both purine and pyrimidine substrates and was maximal with deoxy analogs (dIMP much greater than dUMP greater than dGMP greater than dTMP = dAMP much greater than dCMP) at a pH optimum of 6.2, but slight cross-reactivity occurred with some nondeoxy substrates (IMP greater than GMP greater than UMP = XMP greater than CMP). PyrNase and dNase may be complementary systems that serve physiologically to clear the cytosol of RNA and DNA degradation products during maturation of erythroid elements by conversion of nucleotide monophosphates to diffusible nucleosides.

5'-Nucleotidase↗
PubMed1988

Photosensitizing effects of hematoporphyrin derivative and photofrin II on the plasma membrane enzymes 5'-nucleotidase, Na+K+-ATPase, and Mg2+-ATPase in R3230AC mammary adenocarcinomas.

The sensitivity to photodynamic treatment of three plasma membrane enzymes in R3230AC mammary adenocarcinomas was assessed. The activities of Na+K+-ATPase, Mg2+-ATPase and 5'-nucleotidase in isolated membranes were measured after exposure of membranes to either hematoporphyrin derivative or Photofrin II plus light in vitro or in tumor membranes prepared from animals previously injected with 25 mg/kg Photofrin II and sacrificed at various times prior to exposure to light (in vivo-in vitro protocol). The activities of both Na+K+-ATPase and Mg2+-ATPase were inhibited at equivalent rates by Photofrin II in vitro; inhibition was drug dose and light dose related. For 5'-nucleotidase in vitro, a 10-fold higher porphyrin concentration was required to achieve a similar rate of enzyme inhibition as that for the ion-activated ATPases. Injection of Photofrin II in vivo followed by preparation of tumor plasma membranes, which were subsequently exposed to light in vitro, produced no photosensitization of 5'-nucleotidase activity at any time studied (up to 72 h after Photofrin II administration). Under the same conditions Na+K+-ATPase activity was reduced by 40-60% from 2 to 72 h after drug injection, whereas Mg2+-ATPase activity was inhibited by 10-25% over the same time course. The differential sensitivity of these three enzymes observed in this in vivo-in vitro protocol suggests that each enzyme may possess different characteristics, such as three-dimensional configuration or membrane location, that afford varying susceptibility to porphyrin photosensitization. The data also suggest that photosensitivity-induced damage to these ion-activated plasma membrane ATPases could have deleterious effects on tumor cell survival.

5'-Nucleotidase↗