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PubMed1985

Immunocytochemical localization of 5'-nucleotidase in oligodendroglia and myelinated fibers in the central nervous system of adult and young rats.

Rat central nervous system (CNS) tissue sections were immunostained by the peroxidase-anti-peroxidase (PAP) method using a rabbit serum directed against rat liver 5'-nucleotidase. In paraffin sections from the brains of 60-day-old rats 5'-nucleotidase immunoreactivity occurred in the same white-matter regions as myelin-basic protein immunoreactivity and histological staining of myelin. The immunostaining of cerebral white matter for 5'-nucleotidase was more intense and wide-spread at the age of 120 days than at 60 days, and the choroid plexus and blood vessels were stained consistently. In the paraffin sections from the brains of younger (20-day-old) rats the staining of 5'-nucleotidase in the white matter was faint and patchy. In paraffin sections from spinal cord, 5'-nucleotidase immunoreactivity was observed throughout the lateral white-matter columns and, frequently, in the cell bodies of interfascicular oligodendroglia. Interfascicular oligodendroglia also showed 5'-nucleotidase immunoreactivity in vibratome sections from the CNS tissue of young and adult rats. The findings were consistent with histochemical and biochemical evidence for 5'-nucleotidase in rat brain myelin and oligodendroglia, with substantial increases in activity in the myelin as rats develop from the ages of 20 to 120 days. 5'-Nucleotidase immunoreactivity was not observed in any astrocytes or in oligodendrocytes in the gray matter; however, the enzyme may occur in those glial cells at levels lower than were detectable using the present method.

5'-Nucleotidase↗
PubMed1981

The effects of phospholipids on the properties of hepatic 5'-nucleotidase.

Arrhenius plots of 5'-nucleotidase activity in microsomes or plasma membranes from rat liver exhibited transitions at approximately 35 degrees C. The enzyme was purified from homogenates after solubilization in 2% Triton X-100 and 1% sodium deoxycholate. After the initial steps of the purification, the enzyme was recovered in membranes, as judged by both thin section and freeze-fracture electron microscopy, which contained sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. The purest fractions of 5'-nucleotidase were enriched approximate 3,000-fold, consisted of similar membranes, but only contained sphingomyelin. Thermal transitions were detected in Arrhenius plots of 5'-nucleotidase after detergent solubilization, in the membranes which contained the three phospholipids, but not in the purified fraction which contained only sphingomyelin; transitions were also detected after reassociation of the purified enzyme with microsomal or plasma membrane lipids and phosphatidylcholine but not with phosphatidylethanolamine. Phosphatidylcholines containing specific fatty acids all affected the energy of activation of 5'-nucleotidase, and the detergent Sarkosyl, which has been shown to dissociate phospholipids from 5'-nucleotidase (Evans, W. H., and Gurd, J. W. (1973) Biochem. J. 133, 189-199), caused a marked decrease in the stability of the enzyme to heating. Inhibition of 5'-nucleotidase by concanavalin A followed by reactivation with alpha-methyl-D-mannoside resulted in linear Arrhenius plots of 5'-nucleotidase activity in membrane fractions, and in lower transition temperatures for the detergent, solubilized enzyme. It is concluded that in situ, 5'-nucleotidase interacts with both sphingomyelin and phosphatidylcholine; the first apparently influences the stability of the enzyme and the second, the energy of activation. In addition, the lipid environment of the enzyme seems to be altered as a result of lectin binding.

5'-Nucleotidase↗
PubMed1995

Interaction of singlet oxygen with 5'-nucleotidase in rat hearts.

This study was aimed to determine whether singlet oxygen (1O2) attenuates 5'-nucleotidase activity in the ischemic myocardium. Isolated rat hearts were exposed to either exogenous 1O2 produced by irradiating rose bengal or 40-min ischemia and reperfusion. Ecto-5'-nucleotidase activity was inhibited by exogenous 1O2 (3.74 +/- 0.38 mumol/min/g dry weight), when compared with normal control (7.52 +/- 0.41 mumol/min/g dry weight; P < 0.05). The enzymatic activity was significantly preserved by histidine (25 mM)--a 1O2 scavenger (7.04 +/- 0.61 mumol/min/g dry weight; P < 0.05 v rose bengal group). After ischemia, the activity of ecto-5'-nucleotidase was greatly reduced (2.51 +/- 0.25 mumol/min/g dry weight), when compared with normal control. Histidine significantly enhanced ecto-5'-nucleotidase activity (6.55 +/- 0.52 mumol/min/g dry weight, P < 0.05 v ischemic control). Adenosine release was consistent with ecto-5'-nucleotidase activity. The time course studies of effects of 1O2 on coronary flow, cardiac function, and LDH release revealed that the damage by 1O2 to ecto-5'-nucleotidase activity and adenosine release primarily accounted for impaired coronary flow, cardiac dysfunction, and impaired cardiac metabolism. Lipid peroxidation induced by exogenous 1O2 or ischemia was in parallel with ecto-5'-nucleotidase deactivation by 1O2. It is concluded that 1O2 causes inactivation of ecto-5'-nucleotidase and attenuation of adenosine release which could possibly be one of the important mechanisms of oxygen radical-mediated myocardial injury.

5'-Nucleotidase↗
PubMed1993

A simple assay for ecto-5'-nucleotidase using intact pulmonary artery endothelial cells. Effect of endotoxin-induced cell injury.

Adenosine may be protective in acute vascular injury by inhibiting platelet aggregation and neutrophil oxidant release. In contrast, adenine nucleotides, which may be released with acute vascular injury, stimulate platelet aggregation and neutrophil oxidant release. Ectonucleotidases, membrane enzymes that catabolize extracellular nucleotides, are the primary mechanism for degrading circulating nucleotides to adenosine. Ecto-5'-nucleotidase converts extracellular AMP to adenosine. We hypothesized that endothelial cell injury alters ecto-5'-nucleotidase activity. Using a novel assay first reported by Jamal et al. (Biochem J 250: 369-373, 1988) with rat adipocytes, we studied the properties of ecto-5'-nucleotidase in intact monolayers of cultured bovine pulmonary artery endothelial cells (BPAEC) and examined the effect of endotoxin on enzyme activity. The assay uses a fluorescent analog of AMP, 1,N6-etheno-AMP (E-AMP), as the substrate for ecto-5'-nucleotidase, and measures ethenoadenosine (E-Ado) formation. Etheno-AMP in Hepes buffer, pH 7.4, at 22 degrees, was added to confluent monolayers of BPAEC; samples of supernatant were collected after various intervals, and E-AMP and E-Ado were quantitated by HPLC. Using these methods we found a Km of 15 +/- 6 microM, a pH optimum of 7.48, minimal effect of MgCl2 or CaCl2 at physiologic pH, and inhibition by alpha,beta-methylene ADP, a known 5'-nucleotidase inhibitor. We established that the monolayer assay was indeed measuring cell surface associated 5'-nucleotidase. To determine the effect of endotoxin, we incubated confluent monolayers with endotoxin in Minimal Essential Medium plus 10% fetal bovine serum for 24 hr, washed them, and assessed the conversion of E-AMP to E-Ado by the endotoxin-injured cells. Endotoxin stimulated endothelial ecto-5'-nucleotidase activity. This increase in 5'-nucleotidase activity in response to endotoxin injury may represent an important clearance mechanism for circulating adenine nucleotides and may be protective in acute vascular injury by increasing adenosine production.

5'-Nucleotidase↗
PubMed1983

Properties of 5'-nucleotidase in rat heart sarcolemma.

The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.

Alamethicin↗
PubMed2006

Anticoagulant effect of Naja naja venom 5'nucleotidase: demonstration through the use of novel specific inhibitor, vanillic acid.

The snake venom proteins affect hemostasis by either advancing/delaying blood coagulation. Apart from proteases and phospholipase A(2)s (PLA(2)s), 5'nucleotidase is known to affect hemostasis by inhibiting platelet aggregation. In this study, the possible involvement of Naja naja venom 5'nucleotidase in mediating anticoagulant affect is evaluated. Vanillic acid selectively and specifically inhibited 5'nucleotidase activity among other enzymes present in N. naja venom. It is a competitive inhibitor as evident of inhibition relieving upon increased substrate concentration. Vanillic acid dose dependently inhibited the anticoagulant effect of N. naja venom up to 40%. This partial involvement of 5'nucleotidase in mediating anticoagulant effect is substantiated by concanavalin-A (Con-A) inhibition studies. Con-A, competitively inhibited in vitro protease and 5'nucleotidase activity up to 100%. However, it did not exhibit inhibitory activity on PLA(2). The complete inhibition of anticoagulant effect by Con-A upon recalcification time suggests the participation of both 5'nucleotidase and protease in mediating anticoagulant effect of N. naja venom. Vanillic acid and Con-A inhibition studies together suggest that probably 5'nucleotidase interacts with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Thus, this study for the first time demonstrates the involvement of 5'nucleotidase in mediating N. naja venom anticoagulant effect.

5'-Nucleotidase↗
PubMed1996

A low-Km 5'-nucleotidase from rat brain cytosolic fraction: purification, kinetic properties, and description of regulation by a novel factor that increases sensitivity to inhibition by ATP and ADP.

A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000-g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low Km for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by alpha,beta-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of Mr = 54,300 +/- 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- Km "soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000-g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.

5'-Nucleotidase↗
PubMed1979

Distribution of 5'-nucleotidase in human lymphoid tissues.

Low activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) in T lymphoblasts may explain the marked sensitivity of this cell to deoxynucleotide accumulation when compared to B lymphoblasts. The relevance of such observations with cultured cells to the normal immune system requires the demonstration of similar differences in the 5'-nucleotidase activity of normal human lymphocyte subpopulations. Sheep erythrocyte (E) rosette-forming cells from normal thymus, tonsil, and peripheral mononuclear cells have 5'-nucleotidase activities of 1.7, 11.3, and 21.2 nmol/hr per 10(6) cells. Non-E-rosette forming cells from the peripheral blood or tonsil have 5'-nucleotidase activity comparable to the higher levels found in the peripheral E-RFC. Increased levels of 5'-nucleotidase activity may be a marker for post-thymic T lymphocytes. T lymphoblasts have 5'-nucleotidase activity similar to values demonstrated for E-RFC in thymus, whereas cultured B lymphoblasts have 5'-nucleotidase activity 15 times greater than that of T lymphoblasts. On the basis of these observations, the 5'-nucleotidase deficiency in congenital agammaglobulinemia has been reevaluated. In these patients the data indicate that peripheral E-rosette forming cells have the enzyme deficiency, demonstrating an abnormality of T lymphocytes in this disorder of immunoglobulin production.

Agammaglobulinemia↗
PubMed1991

5'-nucleotidase from the electric ray electric lobe. Primary structure and relation to mammalian and procaryotic enzymes.

A cDNA encoding a 5'-nucleotidase was identified by screening a lambda gt10 cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63,833 Da. The N-terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C-terminus contains a stretch of hydrophobic amino acids, considered to be cleaved on post-translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N-linked glycosylation sites. Electric ray 5'-nucleotidase shares 61% amino acid identity with the enzymes from rat liver and human placenta, and about 23% with bacterial proteins possessing 5'-nucleotidase activity and also additional enzyme activities like UDP-glucose hydrolase. Polyclonal antibodies raised against 5'-nucleotidase from mammalian sources or the electric ray electric organ reveal mutual cross-reactivity. Interestingly, there are 5-7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5'-nucleotidase, 3'-nucleotidase or phosphodiesterase activity. 5'-nucleotidase isolated from Torpedo electric organ hydrolyzes UDP-glucose at 8% of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5'-nucleotidase from multifunctional nucleotide hydrolases is discussed.

5'-Nucleotidase↗
PubMed1996

Role of activation of protein kinase C in the infarct size-limiting effect of ischemic preconditioning through activation of ecto-5'-nucleotidase.

BACKGROUND: We have reported previously that ischemic preconditioning limits infarct size by increasing ecto-5'-nucleotidase activity. Since we have also reported that protein kinase C activation increases ecto-5'-nucleotidase activity in rat cardiomyocytes, we tested whether activation of protein kinase C during ischemic preconditioning contributes to the infarct size-limiting effect through augmentation of ecto-5'-nucleotidase activity in the canine heart. METHODS AND RESULTS: The coronary artery was occluded four times for 5 minutes with alternating 5-minute periods of reperfusion (ischemic preconditioning). Then the coronary artery was occluded for 90 minutes followed by 6 hours of reperfusion. Infarct size, normalized by the risk area, in the ischemic preconditioning group was smaller than in the control group (42.6 +/- 3.6% in the control group versus 7.9 +/- 1.8% in the ischemic preconditioning group, P < .001). Myocardial ecto-5'-nucleotidase activity was increased after the ischemic preconditioning procedure but the increase in ecto-5'-nucleotidase was attenuated by inhibitors of protein kinase C (polymyxin B and GF109203X). Both polymyxin B and GF109203X blunted the infarct size-limiting effect of ischemic preconditioning (infarct size 33.1 +/- 6.9% and 35.1 +/- 6.4%, respectively). The infarct size-limiting effect was also blunted by an inhibitor of ecto-5'-nucleotidase. Transient administration of methoxamine mimicked the increase in ecto-5'-nucleotidase activity and the infarct size-limiting effect, both of which were abolished by inhibitors of protein kinase C. CONCLUSIONS: We conclude that activation of ecto-5'-nucleotidase and protein kinase C contributes to the infarct size-limiting effect of ischemic preconditioning.

5'-Nucleotidase↗
PubMed2006

Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle.

In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57+/-22% of whole homogenate 5'-nucleotidase activity and was not significantly different from the other groups. A weak inverse correlation was found between AMP deaminase and cN-I activities (r2=0.18, p<0.01). There were no significant differences between different groups in the activities of cN-I, whole homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities of whole homogenate nucleotidase, total cN and cN-I. The results indicate no changes in the relative expression or catalytic behaviour of cN-I in AMP deaminase-deficient human skeletal muscle, but suggest that increased turnover of AMP by cN-I in working skeletal muscle is due to higher substrate availability of AMP.

5'-Nucleotidase↗
PubMed1988

A quantitative histochemical study of 5'-nucleotidase activity in rat liver after ischaemia.

The lead salt method of Wachstein and Meisel15 has been applied using incubation media containing polyvinyl alcohol for the localization and quantification of 5'-nucleotidase (E.C.3.1.3.5) activity in cryostat sections from rat liver after ischaemia in vitro and ischaemia in vivo followed by different periods of re-perfusion. 5'-Nucleotidase activity at the bile canaliculi, especially in the pericentral areas, had already decreased after 60 min of ischaemia in vitro, although the total activity as measured densitometrically was not changed. After 120-240 min of ischaemia, a significant decrease of the total 5'-nucleotidase activity was found. At that stage, signs of irreversible cell damage were recognized. Short periods of re-perfusion (1 h) after ischaemia in vivo induced a decreased bile canalicular 5'-nucleotidase activity throughout the entire liver, but a restoration after longer periods of re-perfusion was observed (5, 24, and 48 h). Necrotic areas recognized by a decreased lactate dehydrogenase activity after all periods of re-perfusion showed decreased total 5'-nucleotidase activities. A correlation was observed between the decrease in bile canalicular 5'-nucleotidase activity and the disappearance of microvilli of the bile canaliculi. It is concluded that a decrease in the bile canalicular 5'-nucleotidase activity can be used as a very sensitive marker for ischaemic liver cell damage. Assessment of the irreversibility of the cell injury has to be determined using additional parameters such as a decreased lactate dehydrogenase activity.

5'-Nucleotidase↗
PubMed1987

Purification of 5'-nucleotidase from human placenta after release from plasma membranes by phosphatidylinositol-specific phospholipase C.

5'-Nucleotidase was purified greater than 1000-fold from human placenta by treatment of plasma membranes with S. aureus phosphatidylinositol-specific phospholipase C and affinity chromatography on Con A Sepharose and AMP-Sepharose. The resulting enzyme had a specific activity of greater than 5000 mumol/hr/mg protein and a subunit molecular weight of 73,000. Goat antibodies against 5'-nucleotidase inhibited enzyme activity and detected 5'-nucleotidase after Western blotting. These antibodies also recognized a soluble form of 5'-nucleotidase and residual membrane-bound 5'-nucleotidase which could not be released by phosphatidylinositol-specific phospholipase C treatment, suggesting that the three forms of the enzyme are structurally related. The soluble 5'-nucleotidase may be derived from the membrane-bound form by the action of an endogenous phospholipase C. The structural basis for the inability of some of the membrane-bound 5'-nucleotidase to be released by phosphatidylinositol-specific phospholipase C is unknown.

5'-Nucleotidase↗
PubMed1986

Purification of an enzyme aggregate containing 3',5'-cyclic-nucleotide phosphodiesterase and nucleotidase.

Several steps of purification (octyl-Sepharose chromatography, Blue Sepharose 6B chromatography and sucrose density gradient centrifugation) led to a highly purified aggregate of the enzymes, 3',5'-cyclic-nucleotide phosphodiesterase (PDE) and nucleotidase. The purified enzyme aggregate showed an S value of 7.3 (SE +/- 0.3, n = 10). Further analysis by SDS-polyacrylamide gel electrophoresis (PAGE) revealed two proteins near 67 and 60 kDa. Dissociation of the 7.3 S enzyme aggregate showed a 3.6 S PDE form and a nucleotidase form at 4.2 S. Additionally, higher S value forms of the nucleotidase up to 17 S have been observed. Apparently, they had formed by self-association. SDS-PAGE of the 17 S nucleotidase form showed only one band at 67 kDa. This was taken as evidence for the homogeneity of the 17 S nucleotidase form and the self-association of the nucleotidase after dissociation from the 7.3 S enzyme aggregate. Furthermore, from this it could be concluded that the 67 kDa protein of the 7.3 S enzyme aggregate should be identified with the nucleotidase, and thus the 60 kDa band represents the PDE.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

5'-nucleotidase localization in the brains and spinal cords of adult normal and dysmyelinating mutant (shiverer) mice.

Immunocytochemical staining with the antibody against mouse liver 5'-nucleotidase revealed 5'-nucleotidase antigenicity in myelinated fibers in the brains and in myelinated fibers and some interfascicular oligodendroglia in the spinal cords of normal adult mice. Although the 5'-nucleotidase specific activity in adult shiverer mouse CNS tissue homogenates had been shown to be normal, immunocytochemical staining with anti-mouse-5'-nucleotidase could be demonstrated in CNS tissue sections from only 2 out of 10 of the mutant animals. In tissue from these animals the staining, which was relatively faint, was localized specifically to cell-bodies, usually arranged in rows, and to material oriented parallel to nerve fibers. This pattern of immunostaining with anti-5'-nucleotidase resembled the immunostaining with anti-carbonic anhydrase but not with anti-glial-fibrillary-acidic-protein. This suggested that the rows of cells were oligodendrocytes, not astrocytes, and that the material parallel to nerve fibers might consist of oligodendrocyte processes wrapped loosely around axons. The antibody against rat 5'-nucleotidase, as distinguished from mouse, immunostained only the blood vessels in the shiverer mouse CNS, a finding similar to a previous observation in the normal mouse CNS. From these findings it was inferred that the primary loci of 5'-nucleotidase in the shiverer mouse CNS were interfascicular oligodendrocytes, their processes, and blood vessels, and in the normal mouse CNS, the myelin in some tracts, the blood vessels, and some interfascicular oligodendrocytes.

5'-Nucleotidase↗
PubMed1989

Evidence for the direct interaction of chicken gizzard 5'-nucleotidase with laminin and fibronectin.

The ectoenzyme 5'-nucleotidase purified from chicken gizzard is shown to specifically interact with laminin and fibronectin, components of the extracellular matrix, by a number of different techniques: (i) cosedimentation with laminin by sucrose gradient centrifugation; (ii) affinity adsorption to both laminin- and fibronectin-Sepharose 4-B; (iii) specific binding to both laminin and fibronectin dotted onto cellulose filters; and (iv) monoclonal antibodies against 5'-nucleotidase are shown to interfere with the interaction of 5'-nucleotidase with laminin and fibronectin. For all the techniques employed, the interactions were found to be specific, since 5'-nucleotidase did not bind to unrelated proteins such as bovine serum albumin or to monomeric actin. The interaction of purified chicken gizzard 5'-nucleotidase could be demonstrated for the hydrophobic enzyme solubilized in detergent and after its reconstitution into artificial phospholipid vesicles. The affinity adsorption experiments indicate that reconstituted enzyme binds more strongly to both laminin and fibronectin. The 5'-nucleotidase employed in this study is anchored to the plasma membrane by a glycan-phosphatidylinositol linker. After treatment with phosphatidylinositol-specific phospholipase C, the enzyme is transformed into a hydrophilic form, for which interactions with laminin and fibronectin could also be demonstrated by the dot-blot technique. Thus controlled cleavage of the phosphatidylinositol linker of 5'-nucleotidase could enable cells to rapidly alter their adhesiveness to certain components of the extracellular matrix.

5'-Nucleotidase↗
PubMed1980

Regulation of purine biosynthesis in cultured Drosophila melanogaster cells: II/relationships between hypoxanthine-guanine-phosphoribosyl transferase and 5'-nucleotidase.

All in vitro cell lines from Drosophila melanogaster do not possess measurable hypoxanthine - guanine - phosphoribosyltransferase (HGPRT) and 5'-nucleotidase activities. Nevertheless these enzymatic activities could be detected by the modification of culture conditions or treatment of crude extracts from Drosophila cells (1). A Drosophila melanogaster hemocyte line enabled demonstration of a relationship between the activities of HGPRT and 5'-nucleotidase. Only the addition of azaserine to the culture medium allows detection of a measurable amount of HGPRT and 5'-nucleotidase in cells devoid of endogenous synthesis of purine nucleotides. This action of azaserine suggests that the simultaneous expression of both enzymes is first directed by the suppression of the 5'-nucleotidase inhibition by pyrimidine nucleotides, and later on by the 5'-nucleotidase activity hydrolyzing the mononucleotides which inhibited the HGPRT, resulting its expression. This regulation system is confirmed by the simultaneous recovery of HGPRT and 5'-nucleotidase activities after heating cell cultures or cell extracts. Under such experimental conditions, 5'-nucleotidase is desensitized towards its negative effectors, while HGPRT regulation remains unchanged.

5'-Nucleotidase↗
PubMed1987

Reconstitution of purified chicken gizzard 5'-nucleotidase in phospholipid vesicles. Evidence for its transmembraneous character and the existence of functional domains on both sides of the phospholipid bilayer.

5'-Nucleotidase, purified to homogeneity from chicken gizzard using published procedures [Dieckhoff, J., Knebel, H., Heidemann, M. and Mannherz, H. G. (1985) Eur. J. Biochem. 151, 377-383] was incorporated into artificial phospholipid vesicles after prolonged dialysis against detergent-free buffer or by a gel filtration procedure. After dialysis the obtained liposomes exhibit a mean diameter of 80 nm and contain 5'-nucleotidase at random orientation, demonstrated by finding up to 50% of the total liposome-incorporated AMPase activity to be cryptic, i.e. could only be measured after their permeabilization by addition of detergent. By affinity chromatography a phospholipid vesicle fraction could be obtained containing almost exclusively cryptic AMPase activity, thus representing the inside-out orientation of 5'-nucleotidase. Comparative analysis of physiochemical and enzymatic properties of 5'-nucleotidase reveals differences between the detergent-solubilized and the liposome-incorporated 5'-nucleotidase including a changed accessibility of the enzyme to polyclonal and monoclonal antibodies. Binding and AMPase inhibition studies with different polyclonal antibodies strongly indicate to the existence of a cytoplasmic domain of chicken gizzard 5'-nucleotidase. F-actin appears preferentially to interact with the cytoplasmic domain of liposome-incorporated 5'-nucleotidase.

5'-Nucleotidase↗