Activity of cytosolic 5'-nucleotidase in regenerating rat liver after partial hepatectomy

The specific activity of the cytosolic 5'-nucleotidase in regenerating liver increased to 175% of the control level of sham-operated animals during the 2nd and 3rd day and remained elevated most of the experimental period. The total cytosolic 5'-nucleotidase activity of the regenerating liver reached the level of control rats between 2 and 3 days after the operation. The variation pattern of the enzyme, which was distinctly different from variations of other known phosphohydrolases, was strikingly similar to that of the salvage enzyme hypoxanthine/guanine phosphoribosyltransferase.     

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.     

Target size of 5'-nucleotidase in smooth muscle

Target size of the 5'-nucleotidase in six different smooth muscles was determined by radiation inactivation. The enzyme in the soluble fraction of rat myometrium and vas deferens gave a target size of approximately 80,000 daltons. The plasma membrane bound 5'-nucleotidase however, gave target size of 80,000 to 110,000 daltons in rat gastric fundus and vas deferens and dog stomach and ileum, 135,000 daltons in rat mesenteric artery and 210,000 daltons in rat myometrium.     

5'-nucleotidase from bull seminal plasma

5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) occurs in bull seminal plasma in multiple forms. The heterogeneity does not reflect the existence of true isoenzymes, but is due to the association of the enzyme with particulate material and to molecular aggregation phenomena. Addition of detergents to native bull seminal plasma prevents molecular aggregation, solubilizes the particulate form of the enzyme, and results in the appearance of a single molecular form of the enzyme. Enzyme purification can be achieved after three chromatographic steps which involve negative adsorption of 5'-nucleotidase activity on DEAE-Sephadex A-50 followed by two affinity chromatographies on concanavalin A-Sepharose 4B and ADP-agarose. The enzyme appears to be a dimeric glycoprotein. Some properties of the enzyme, including substrate specificity and the effects of hydrogen ion concentration and of various divalent cations, are reported.     

Diadenosine tetraphosphate activates cytosol 5'-nucleotidase

The rate of hydrolysis of IMP (0.5 mM) by cytosol 5'-nucleotidase from Artemia embryos was increased up to 7-fold by concentrations of around 10 microM diadenosine tetraphosphate (Ap4A). Half maximal activation of the enzyme was accomplished with 5 microM Ap4A. The Km (S 0.5) values of the nucleotidase for IMP, GMP, AMP, XMP and CMP decreased about 10 fold in the presence of 10 microM Ap4A. Maximum velocity of the enzyme was not affected by Ap4A. ATP had been previously described as an activator of the enzyme. However, comparatively with Ap4A, concentrations of ATP two orders of magnitude higher are needed to elicit similar effects on the enzyme. Preliminary results indicate that Ap4A is also an activator of the cytosol 5'-nucleotidase from rat liver.     

Cytoplasmic 5'-nucleotidase catalyzes acyclovir phosphorylation

A cytoplasmic 5'-nucleotidase (EC 3.1.3.5) can catalyze the phosphorylation of inosine (Worku, Y., and Newby, A.C. (1982) Biochem. J. 205, 503-510). This enzyme was purified to determine whether it could catalyze the formation of trace levels of phosphorylated acyclovir (ACV), a nucleoside analog with antiherpes activity. Acyclovir phosphorylating activity from rat liver co-chromatographed with the enzyme throughout the 1200-fold purification and through size exclusion chromatography or polyacrylamide-gel electrophoresis. In addition, the pH optimum, ATP stimulation, and phosphate inhibition of the ACV phosphorylating activity paralleled those of the 5'-nucleotidase. Finally, ACV phosphorylation was competitively inhibited by inosine (Kis = 6.5 mM; K'm (inosine) = 5.0 mM). This was consistent with phosphorylation at a common catalytic site. In addition to inosine and ACV, the guanine derivatives Guo, dGuo, 9-beta-D-arabinofuranosylguanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were substrates for the enzyme. The relative phosphorylation rates were, respectively, 100, 0.7, 19, 4, 0.3, and 0.7, at 0.1 mM phosphate acceptor. Approximate K'm values were, respectively, 5, 90, 10, 10, greater than 100, and greater than 100 mM. Although the substrate activity of ACV with the 5'-nucleotidase was inefficient, it appeared to be sufficient to account for the small amounts of ACV phosphates formed in uninfected cells.     

Age-dependency of lymphocyte ecto-5'-nucleotidase activity

The activity of lymphocyte ecto-5'-nucleotidase (ecto-5'-NT) decreases with advancing age, T lymphocyte ecto-5'-NT activity begins to fall after the age of 40 and subjects in the 41 to 50, 51 to 60, 61 to 75, and 75 to 85 age ranges of life have 57, 52, 38, and 19%, respectively, of the T lymphocyte ecto-5'-NT activity of subjects under the age of 40. TG cells (suppressor cells) have 39% of the ecto-5'-NT activity of Tnon-G cells (helper cells) but the increase in numbers of TG cells that occurs with age explains only about 14% of the age-related fall in T lymphocyte ecto-5'-NT activity. B lymphocyte ecto-5'-NT activity remains stable until age 60 and subjects over 60 years of age have 42% of the B lymphocyte ecto-5'-NT activity of subjects under age 60.     

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.     

Absence of 5'-nucleotidase in porcine polymorphonuclear leucocyte membranes

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess p-nitrophenyl phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5'-nucleotidase inhibitor alpha, beta-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5'-nucleotidase. An antibody against mouse liver 5'-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5'-nucleotidase and the necessity for distinguishing between true 5'-nucleotidase and non-specific phosphatase activity is discussed.     

Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.