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PubMed1983

5'-Nucleotidase is activated upon cholesterol-depletion of liver plasma membranes.

The cholesterol content of rat liver plasma membranes was manipulated using either cholesterol-free or cholesterol-enriched liposomes. Removal of cholesterol from the membranes led to a marked increase in 5'-nucleotidase activity. However, increase in cholesterol content failed to exert any significant effect on 5'-nucleotidase activity. Arrhenius plots of the activity of the native enzyme exhibited a break at around 28 degrees C with the activation energy of the reaction less above this temperature than below. In cholesterol-depleted membranes a single break at around 26 degrees C was observed with activation energies greater above this temperature than below it. In cholesterol-enriched membranes Arrhenius plots were linear over the range examined. It is suggested that the lipid environment of the external half of the bilayer only influences 5'-nucleotidase activity in these membranes and that cholesterol exerts controlling effects on both the activity and conformation of the enzyme in native membranes.

5'-Nucleotidase↗
PubMed1984

Identification of histidyl and cysteinyl residues essential for catalysis by 5'-nucleotidase.

Inactivation of both cytosolic 5'-nucleotidase and ecto-5'-nucleotidase by diethylpyrocarbonate indicated the presence of an essential histidyl residue which in the cytosolic enzyme was conclusively located at the active site. Inactivation by thiol reagents indicated the presence of an essential cysteinyl residue in both enzymes. The data suggest that both 5'-nucleotidases belong to a group of histidine phosphatases which also includes glucose-6-phosphatase and acid phosphatase. A working hypothesis for the catalytic mechanism of these enzymes is proposed.

5'-Nucleotidase↗
PubMed1986

The extracellular matrix proteins laminin and fibronectin modify the AMPase activity of 5'-nucleotidase from chicken gizzard smooth muscle.

Laminin and fibronectin, but not collagen, affect the AMPase activity of the purified transmembrane protein 5'-nucleotidase. Laminin stimulates whereas fibronectin inhibits the AMPase activity of this ectoenzyme. The AMPase-modulating effects by these components of the extracellular matrix require a preincubation period of several hours when detergent-solubilized 5'-nucleotidase is employed, they can, however, instantaneously be elicited with liposome-incorporated 5'-nucleotidase.

5'-Nucleotidase↗
PubMed1983

Developmental alterations in 5'-nucleotidase kinetics and lipid composition of rat heart sarcolemma.

The objective of these studies was to examine the mechanism by which the specific activity of heart sarcolemma 5'-nucleotidase decreases as function of age. We examined the kinetic properties and the lipid composition of the sarcolemma from animals with different ages. The age groups used were 1 month, 6-8 months and 13-15 months. It was found that the Km of this enzyme increases as the animal develops from 1 month to 6-15 months. The opposite was true with 5'-nucleotidase Vmax. There was no significant difference between the middle age and the older age groups in those parameters. The results of these experiments suggest that the increase in Km in sarcolemma 5'-nucleotidase could be due to the reduction of the sarcolemmal polyunsaturated fatty acid concentration, the only lipid alteration observed.

5'-Nucleotidase↗
PubMed1985

Parasite-specific inhibition of 5'-nucleotidase from Onchocerca volvulus and Dirofilaria immitis by the amoscanate-derivative CGP 8065.

The presence of 5'-nucleotidase was demonstrated in Onchocerca volvulus and Dirofilaria immitis; the bulk of activity was found in the particulate fraction. The enzyme of filarial worms exhibited a broad pH-optimum between 6.4 and 8.0 and substrate specificity for nucleotides compared to glucose-6-phosphate and p-nitrophenyl phosphate. The apparent Km-values for AMP were found to be 0.15 mM and 0.22 mM for the enzyme from O. volvulus and D. immitis, respectively. The activity of 5'-nucleotidase from both filarial worms was effectively inhibited by the filaricidal compound CGP 8065, a dithiocarbamate-derivative of amoscanate, whereas the 5'-nucleotidase from rat liver was not affected. The parasite-specific inhibition by CGP 8065 was found to be reversible and to be competitive with respect to the substrate AMP. The inhibition constants were calculated to be 24 microM and 8 microM for the enzyme from O. volvulus and D. immitis, respectively.

5'-Nucleotidase↗
PubMed1989

Structural comparison of the 68 kDa laminin-binding protein and 5'-nucleotidase from chicken muscular sources: evidence against a gross structural similarity of both proteins.

The 68 kDa laminin-binding protein purified from chicken skeletal muscle and the ectoenzyme 5'-nucleotidase from chicken gizzard are both able to interact with laminin. They were both shown to possess a nearly identical amino acid composition. The 79 kDa glycosylated form of 5'-nucleotidase can be transformed into an enzymatically active form by treatment with endoglycosidase F (Endo F). Deglycosylated (Endo F-treated) 5'-nucleotidase exhibits an apparent molecular mass of 68 kDa. Using immunological and finger-printing techniques, both proteins were analysed to determine their structural relatedness. The results obtained indicate that both proteins are not identical but may posses a few common peptides of yet unknown sequence and length.

5'-Nucleotidase↗
PubMed1984

Thyrotropin increases 5'-nucleotidase activity in primary cultures of porcine thyroid cells.

Thyrotropin given to support-anchored cultures of porcine thyroid cells, either in a serum-containing or in a serum-free system, produced an increase of about 50% in the total activity of 5'-nucleotidase. In the serum-free culture, in which TSH was administered to well-reformed follicles, this increase in 5'-nucleotidase activity concerns both the ecto-enzymic and intracellular forms of the enzyme and it coincides with the period of several days during which several glycosyltransferase activities are elevated and thyroglobulin production increased. Taken together, and in view of a recent in vitro study (Brandan and Fleisher, 1982) documenting the fate of uridine diphosphate in Golgi vesicles, these results suggest that there might be a functional correlation between the stimulation of 5'-nucleotidase and an increased production of nucleoside mono- and diphosphates when the activity of a number of glycosyltransferases is increased.

5'-Nucleotidase↗
PubMed1988

Histochemical localization of 5'-nucleotidase in the reeler mutant mouse.

In the normal mouse cerebellum, 5'-nucleotidase localization exhibits a parasagittal organization with bands of positive and negative staining within the molecular layer. The reeler cerebellum, which possesses an abnormal cytoarchitecture with numerous ectopically located Purkinje cells, was stained histochemically for the presence of 5'-nucleotidase. The resulting staining pattern suggests that 5'-nucleotidase is found in association with areas containing subpopulations of Purkinje cells, independent of whether they are in normal or ectopic positions.

5'-Nucleotidase↗
PubMed1986

Differential composition of cytosol 5'-nucleotidases between T and B lymphoblasts.

WI-L2 cells (a B-lymphoblastoid cell line) were more resistant than CEM cells (a T-lymphoblastoid cell line) to deoxyadenosine, ara-A (9-beta-D-arabinofuranosyladenine), or ara-C (1-beta-D-arabinofuranosylcytosine) inhibition. This was caused by a difference in the composition of cytosol 5'-nucleotidases between WI-L2 and CEM cells. In intact cells, the endogenous production of deoxyadenosine from WI-L2 cells deficient in adenosine kinase (EC 2.7.1.20) and deoxycytidine kinase (EC 2.7.1.74) was consistently high, despite changes in endogenous adenosine production. Endogenous production of deoxyadenosine from CEM cells deficient in adenosine kinase and deoxycytidine kinase was, however, coordinated with endogenous adenosine production. In broken cells, cytosol dAMPase (2'-deoxyadenosine 5'-monophosphate 5'-nucleotidase) activity of WI-L2 cells was 3-5-fold higher than that of CEM cells. dAMPase activity could be separated from ATP-activated IMPase (inosine 5'-monophosphate 5'-nucleotidase) by gel filtration (molecular weight: dAMPase; 39,000-46,000; ATP-activated IMPase, greater than 150,000). Cytosol ATP-activated IMPase and dAMPase were isolated by phosphocellulose or DEAE-Bio-Gel A chromatography from non-specific phosphatases. The ATP-activated IMPase showed only marginal activity towards dAMP (2'-deoxyadenosine 5'-monophosphate), ara-AMP (9-beta-D-arabinofuranosyladenine 5'-monophosphate), or ara-CMP (cytosine-beta-D-arabinofuranoside 5'-monophosphate), even in the presence of ATP. The activity of ATP-activated IMPase was similar in WI-L2 and CEM cells. dAMPase was separated into two peaks by DEAE-Bio-Gel A chromatography; one of these peaks degraded ara-AMP and ara-CMP. The activities of both peaks from WI-L2 cells were higher than those from CEM cells. These results show that the degradation of dAMP, ara-AMP or ara-CMP was more specific and rapid in WI-L2 than in CEM cells.

5'-Nucleotidase↗
PubMed1983

Hepatic plasma membranes from genetically obese (ob/ob) mice: studies on fluorescence polarization, phospholipid composition and 5'-nucleotidase activity.

Hepatic plasma membrane lipids of lean (+/?) and obese (ob/ob) mice have been investigated using 1,6-diphenylhexatriene (DPH). Arrhenius plots of DPH fluorescence polarization in membranes showed the breakpoint in obese mice was reduced from 21 to 15 degrees C, whereas the breakpoint of 5'-nucleotidase activity was raised from 23 to 32 degrees C. Arrhenius break temperatures of DPH polarization and 5'-nucleotidase activity responded differently to housing mice at 34 degrees C and triiodothyronine (T3) treatment. Studies of DPH polarization in liposomes and phospholipid fatty acid composition suggested that differences in sphingomyelin acyl composition determine Arrhenius characteristics of hepatic 5'-nucleotidase in lean and obese mice.

5'-Nucleotidase↗
PubMed1984

Cytochemical localization of 5'-nucleotidase activity in retinal photoreceptor cells.

Further cytochemical studies on the ultrastructural localization of 5'-nucleotidase in the rat retina have revealed activity to be associated with the complex synapses formed by the rod spherules of the receptors and the bipolar and horizontal cell processes. Activity was also seen on the axolemma of receptor fibers. In the rod inner segment strong reaction product is located intracellularly. In the rod outer segment the enzyme appears to be located only on the cytoplasmic side of the disc membrane and not intradiscally . Retinal pigment cells are rich in 5'-nucleotidase. Their microvilli accompany the tips of the receptor cells and show enzyme activity in an ecto position. A role for 5'-nucleotidase is possible in the metabolism of guanylate and adenylate nucleotides both of which are important for visual transduction processes.

5'-Nucleotidase↗
PubMed1987

Detection of ecto-5'-nucleotidase on rat B and T lymphocyte subpopulations using a monoclonal antibody in rosetting reactions.

A mouse monoclonal antibody to rat 5'-nucleotidase (5N 4-2 McAb) was used in the direct anti-determinant rosetting reaction (DARR) to demonstrate the ecto-5'-nucleotidase molecule in preparations of rat lymphocytes. Results indicated that 35.5 +/- 7.5% of peripheral blood lymphocytes (PBL), 37.3 +/- 4.8% of lymph node cells (LN) and 37.0 +/- 8.5% of spleen lymphocytes expressed the 5N 4-2 antigen. Depletion studies and mixed rosetting reactions (MRR) showed that the 5N 4-2 antigen was mainly expressed on rat T lymphocytes rather than on B lymphocytes: In fact 59.6 +/- 3.2% (in PBL), 76.5 +/- 0.6% (in LN) and 67.1 +/- 1.3% (in spleen) of T lymphocytes exhibited the 5N 4-2 antigen compared to only 26.5 +/- 2.6% (in PBL), 34.0 +/- 2.1% (in LN) and 46.1 +/- 12.0% (in spleen) of B lymphocytes. As expected a strong association was found between the expression of 5N 4-2 antigen and 5'-nucleotidase enzyme activity on lymphocytes. Both 5N 4-2 positive cells and enzyme activity were preferentially exhibited in the T lymphocyte subpopulation, and 92% of the enzyme activity was observed in a 5N 4-2 antigen positive subpopulation.

5'-Nucleotidase↗
PubMed1984

Development and properties of a monoclonal antibody specific for human ecto-5'-nucleotidase.

After immunization with purified human placental ecto-5'-nucleotidase and fusion, 18 different hybrids were obtained which produce an antibody inhibitory for enzyme activity. These antibodies show complete cross-reactivity with the enzyme in a membrane-bound form or on the surface of intact cells. After cloning and ascites production, the antibody of one clone ( IFH - 5N1 ) was studied in greater detail. The IFH - 5N1 antibody is of the IgG 1 subclass. Optimal enzyme inhibition is 90% for purified 5'-nucleotidase and 80% for the enzyme on lymphoblasts. The specificity of this antibody is further demonstrated by enzyme inhibition assays and fluorescence labeling using various 5'-nucleotidase-positive and -negative human cells such as peripheral blood lymphocytes, leukemic cells, lymphoblastoid B- and T-cell-lines and fibroblasts. The antibody should provide a useful tool for the diagnosis of certain forms of acute leukemias and for the study of normal human lymphocyte subpopulations.

5'-Nucleotidase↗
PubMed1986

Relative activities of 5'-nucleotidase and adenosine deaminase in atrial and ventricular myocardium--the enzyme paradox.

The 5'-nucleotidase and adenosine deaminase activities were determined for tissue extracts of up to 10 defined regions of the normal dog heart. The specific activity of 5'-nucleotidase was very significantly higher in atrial than in ventricular myocardium (4 X) and was also higher in right than in left sites of the heart (2 X). The reason for the markedly higher specific activity in atrial muscle is not clear and appears paradoxical relative to adenine nucleotide content and pattern of ATP decay during oxygen deprivation. In contrast to 5'-nucleotidase activity the total adenosine deaminase activity was found to be similar in all sampling sites examined.

5'-Nucleotidase↗
PubMed1982

Orientation and role of nucleosidediphosphatase and 5'-nucleotidase in Golgi vesicles from rat liver.

The fate of UDP formed during the galactosylation of added N-acetylglucosamine in Golgi vesicles isolated from rat liver using D2O-sucrose gradients has been determined. UDP-Gal labeled with [14C]uracil was used, and the products of the reaction were separated and quantitated by using high-pressure liquid chromatography. [14C]Uridine rather than [14C]UDP or [14C]UMP was found to accumulate, indicating the presence of both UDPase and UMPase activities in the Golgi. Golgi vesicles were shown to contain a nucleosidediphosphatase activity that is membrane bound. It appears to be located on the luminal face of the Golgi since it is activated 3-5-fold by detergents and 4-fold by treatment of the vesicles with Filipin. We have shown previously that Filipin disrupts the Golgi but does not solubilize membrane-bound enzymes. The nucleosidediphosphatase of the Golgi differs from that present in rough endoplasmic reticulum in its absolute requirement for Ca2+ for activity and in its substrate specificity that is higher for UDP than for IDP. Golgi vesicles also contain UMPase activity that is stimulated only 2-fold by detergents or Filipin. Concanavalin A inhibits this activity about 80% in both intact and detergent-treated vesicles. The Golgi UMPase is thus probably identical with 5'-nucleotidase. These results are consistent with histochemical evidence from other laboratories that indicate that 5'-nucleotidase is present on both sides of liver Golgi membranes. In the presence of concanavalin A and N-acetylglucosamine, intact Golgi vesicles were found to convert UDP-Gal to UMP. These findings indicate that UDP formed by galactosyltransferase in the lumen of the vesicles is rapidly converted to UMP by UDPase in the lumen but that UMP moves rapidly out of the lumen of the Golgi and is broken down to uridine by 5'-nucleotidase on the cytoplasmic side of the vesicles.

5'-Nucleotidase↗
PubMed1980

Stereochemistry of the hydrolysis of adenosine 5'-thiophosphate catalyzed by venom 5'-nucleotidase.

The stereochemical problem involving a pro-pro-prochiral phosphorus center, the hydrolysis of adenosine 5'-monophosphate to adenosine and inorganic phosphate catalyzed by the venom 5'-nucleotidase, has been studied by use of chiral [16O, 17O, 18O]thiophosphates (Psi). (Rp)- and (Sp)-[alpha-18O1]Adenosine 5'-thiophosphates (AMPS) were synthesized by a combined chemical and biochemical procedure. Hydrolysis of (Rp)- and (Sp)-[alpha-18O1]AMPS in H217O by 5'-nucleotidase gave two enantiomers of chiral Psi of unknown configuration. A 31P NMR method based on the combination of the quadrupolar effect of 17O [Tsai, M.-D. (1979) Biochemistry 18, 1468-1472] and the 18O isotope shift [Cohn, M., & Hu. A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 200-203] has been developed to analyze the configuration of chiral Pso. The results indicate that hydrolysis of (Rp)- and (Sp)-[alpha-18O1]AMPS in H217O gave (R)- and (S)- [16O, 17O, 18O]Psi, respectively. Therefore the hydrolysis of AMPS catalyze by the venom 5'-nucleotidase must proceed with inversion of configuration at phosphorus, which suggests that the reaction is most likely an "in line" single displacement without involving a phosphoryl-enzyme intermediate and without pseudorotation.

5'-Nucleotidase↗
PubMed1986

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5'-nucleotidase.

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.

4-Nitrophenylphosphatase↗