Ecto-5'-nucleotidase activity in T and B lymphocytes from normal subjects and patients with congenital X-linked agammaglobulinemia.

Ecto-5'-nucleotidase activity was measured in lymphocyte subpopulations isolated from normal subjects and patients with congenital X-linked agammaglobulinemia. B lymphocytes from normal subjects have at least three times more ecto-5'-nucleotidase activity than T lymphocytes. Patients with X-linked agammaglobulinemia have 56% of normal activity in their T cells, and lack a lymphocyte subpopulation high in nucleotidase activity. High activity of ecto-5'-nucleotidase may be a biochemical marker for mature surface immunoglobulin-bearing B cells.     

Regional and Subcellular Distribution in Mammalian Brain of the Enzymes Producing Adenosine

The activities of 5'-nucleotidase, 2'-nucleotidase, alkaline phosphatase, and acid phosphatase were measured in rat and autopsied human brains. The four phosphatases in the rat brain showed little change in activity after death. The activities of adenosine-producing enzymes were compared in various parts of rat and human brains. When phosphatase activity was measured at pH 7.5, 5'-nucleotidase showed the highest activity in the most parts of the brain. The activity of 2'-nucleotidase and that of nonspecific phosphatase were almost the same at pH 7.5. However, higher phosphatase activity was observed in all parts of the brain when nonspecific phosphatase activity was measured at pH 10.0 or 5.5. High specific activity of 5'-nucleotidase in the brain was detected in the membranous components, especially in the synaptic membranes. The activity of 2'-nucleotidase was distributed in the soluble and synaptosomal fractions. The highest activity of both alkaline and acid phosphatases was recovered in the crude mitochondrial fraction, with the highest specific activity in the microsomal fraction. Phosphatase activity was distributed widely in the rat brain. The activity of 5'-nucleotidase was high in the medulla oblongata, thalamus, and hippocampus, but low in the peripheral nerve, spinal cord, and occipital lobe. The activity of 2'-nucleotidase was high in the vermis and frontal lobe. The highest acid and alkaline phosphatase activities were detected in the frontal lobe and in the olfactory bulb, respectively. The distribution of the four phosphatases in the autopsied human brain was similar to that in the rat brain. The highest 5'-nucleotidase activity was observed in the temporal lobe and thalamus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular distribution and movement of 5''-nucleotidase in rat cells.

1. Cell-surface 5'-nucleotidase was assayed by incubating whole-cell suspensions with 5'[3H]-AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell-surface 5'-nucleotidase in hepatocytes, adipocytes and lymphocytes isolated from the rat was 15.0, 0.5 and 0.8pmol/min per cell at 37 degrees C respectively. 2. Disruption of the cells by vigorous mechanical homogenization or detergent treatment exposed additional 5'-nucleotidase activity, which represented 52%, 25% and 21% of the total activity in the three cell types respectively. This increase in 5'-nucleotidase activity which occurred when the cells were homogenized was due to a second pool of 5'-nucleotidase within the cell, rather than activation of the cell-surface enzyme. 3. In hepatocytes the intracellular 5'-nucleotidase activity was membrane-bound, indistinguishable from cell-surface 5'-nucleotidase in its inhibition by rabbit anti-(rat liver 5'-nucleotidase) serum and its kinetics with AMP, and was located on the extracytoplasmic face of vesicles within the cell. 4. The cell-surface 5'-nucleotidase of rat hepatocytes was rapidly inhibited when rabbit anti-(rat liver 5'-nucleotidase) serum or concanavalin A was added to the medium at 37 degrees C. Incubation with antiserum for 5 min at 37 degrees C inhibited 83 +/- 3% of the cell-surface enzyme. 5. Incubation of hepatocytes with exogenous antiserum or concanavalin A for 30 min at 37 degrees C resulted in over 50% inhibition of the intracellular enzyme. This inhibition was not prevented by disruption of the cytoskeleton or by ATP depletion. 6. Incubation of hepatocytes with exogenous antiserum or concanavalin A for up to 2h at 0 degrees C caused little or no inhibition of the intracellular enzyme, but over 75% inhibition of the cell-surface enzyme. 7. When surface-inhibited hepatocytes were washed and resuspended in buffer at 37 degrees C, 5'-nucleotidase was observed to redistribute from the intracellular pool to the cell surface.     

Variations in 5'-nucleotidase activity in established mesenchymal cell lines from syngeneic A/J mice

5'-Nucleotidase activity was analyzed in four different mesenchymal cell lines (F, m, e and SP) established from syngeneic A/J mice. The 5'-nucleotidase activity of fibroblasts was lower in transformed cells (F and m) than in nontransformed cells (e). An increase in cell contact during confluence or during high cell density increased 5'-nucleotidase activity, and a decrease in cell contact caused a decrease in 5'-nucleotidase activity in both fibroblastic (F, m and e) and reticulum (SP) cell lines. These results are evidence that 5'-nucleotidase activity in mesenchymal cells is influenced by intercellular contact as well as transformation.     

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.     

Alterations in hepatic 5'-nucleotidase in the tumor-bearing rat

The effect of the growth of the Walker 256 carcinoma on the level of 5'-nucleotidase and alkaline phosphatase in the whole liver and in an isolated hepatocyte membrane preparation of its host was investigated. Alkaline phosphatase activities of whole liver and plasma membrane were increased approximately 5-fold by tumor growth. A 50% decrease in whole liver 5'-nucleotidase activity was observed in tumor-bearing rats while the 5'-nucleotidase activity per milligram membrane protein was unaltered. Tumor growth would therefore appear to affect a pool of 5'-nucleotidase which is not associated with the plasma membrane.     

Activity of cell-detached 5'-nucleotidase in thymocytes of various densities

The rat thymocytes submitted to heating at 45 degrees C for 1 hr liberate plasma membrane fragments containing 5'-nucleotidase activity in the supernatant. The thymocytes were separated by ficoll density gradient centrifugation. High activity of 5'-nucleotidase per 10(6) cells was found in the supernatant of low density (1.069) subset of thymocytes. Thymocyte supernatant of rats treated with hydrocortisone demonstrated higher 5'-nucleotidase activity per 10(6) cells than in intact animals. This is due to an increase of the low density population of thymocytes in treated rats since the 5'-nucleotidase activity per 10(6) cells of the supernatant obtained from this density fraction is the same both in treated with hydrocortisone and intact rats. Hydrocortisone seems to induce a selection of the thymocytes with high 5'-nucleotidase activity.     

5''-Nucleotidase in Rat Brain Myelin

Rat brain myelin showed substantial activity of 5'-nucleotidase. The specific activity in myelin was enriched two- to threefold over that in rat brain homogenates, and the total activity in myelin accounted for approximately 24% of the activity in the homogenates. The 5'-nucleotidase in the homogenates and in isolated myelin had optimum activity at pH 7.5--9.0, was stimulated by Mg2+ and Mn2+, and was inhibited by Co2+, Zn2+, EDTA, and EGTA. 5'-AMP, 5'-UMP, and 5'-CMP were the preferred substrates, and 5'-GMP was hydrolyzed at approximately one-half the rate of the other mononucleotides. The very low rates of cleavage of beta-glycerophosphate and 2'-AMP ruled out any significant contribution of nonspecific phosphatase to the observed 5'-nucleotidase activity in myelin. The 5'-nucleotidase was inhibited by concanavalin A and was protected by alpha-methyl-D-mannoside against inhibited by that lectin, suggesting that this enzyme in the CNS is a glycoprotein. It is concluded from these data, and from histochemical observations made in other laboratories, that the myelin sheath is one major locus of 5'-nucleotidase in the rat brain.     

The synthesis and turnover of 5'-nucleotidase in primary cultured hepatocytes

The synthesis and degradation of 5'-nucleotidase has been studied in rat hepatocytes. Primary cultures of rat hepatocytes were established with the cells showing evidence of polarity after 24-36 h in culture. After a 30 h lag period 5'-nucleotidase activity increased to a plateau level similar to the activity found in whole liver. The half life of the enzyme after reaching the plateau of activity was 22.8 h. Pulse-chase biosynthetic labelling studies of 5'-nucleotidase in the cultured hepatocytes using [35S]methionine showed that the 5'-nucleotidase monomer was synthesised as an Mr 67,000 form which was converted to the mature Mr 72,000 form. [35S]Methionine labelling studies in the presence of tunicamycin showed that the unglycosylated protein monomer was an Mr 57,000 form. The immature Mr 67,000 form of 5'-nucleotidase was sensitive to endoglycosidase H, whereas the mature form was sensitive only to endoglycosidase F. The data presented are consistent with 5'-nucleotidase in a polarised cell being synthesised and processed like other membrane glycoproteins, in contrast to earlier reports.     

Enhancement of 5'-nucleotidase activity of human leukemic cells after fractionation: implications for cancer and aging

Previous studies reported that 5'-nucleotidase activity was undetectable or at much lower levels in the homogenate of human chronic lymphocytic leukemic (CCL) cells than in normal lymphocytes. In the present study, 5'-nucleotidase specific activity in acute myelocytic leukemia (AML), which varied in a range from undetectable to 1.4 (nmoles/min.mg protein), was enhanced by cell fractionation, from undetectable in the homogenate, up to 18.8 +/- 1.2, 6.4 +/- 0.7 and 0.68 +/- 0.12 in plasma membranes, microsomes, and cytosol fraction, respectively. In a further fractionation of the cytosol of various leukemic cells with ammonium sulfate, 5'-nucleotidase specific activity increased up to 14-fold in the 60% (NH4)2SO4 fraction, with a recovery of 1266 +/- 115%. These data suggest that 5'-nucleotidase activity in fractionated leukemic cells is higher than reported previously and that the sum of 5'-nucleotidase activity in subcellular compartments is higher than that detected in the homogenate. Furthermore, even when 5'-nucleotidase was undetectable in a homogenate, it became detectable in the plasma membranes, suggesting that its ecto-enzyme function is still active in leukemic cells. The undetectable or low 5'-nucleotidase in the homogenate is indicative of (1) the enzyme itself being in an inactive form but becoming active after the fractionations, or (2) the presence of a factor(s) that prevents the enzyme from being detected but that is separated from the enzyme by the fractionations. In both cases, the rate of nucleotide catabolism by inactive 5'-nucleotidase in rapidly proliferating leukemic cells should be slower than when the enzyme is active. The present finding is consistent with our previous findings that during normal cell aging the high 5'-nucleotidase activity is associated with senescent non-proliferating cells but low or undetectable activity with rapidly proliferating immortal cells. The implications of 5'-nucleotidase for DNA synthesis in aging and cancer are discussed.     

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.     

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.     

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.     

Diurnal variation in 5'-nucleotidase activity in rat liver. A quantitative histochemical study

The diurnal variation of 5'-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5'-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5'-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5'-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.     

Distribution of 5'-nucleotidase in muscle of some marine fishes.

A preliminary examination for the purification and characterization of 5'-nucleotidase of fish muscle was carried out and the following results were obtained. 1. The activities of 5'-nucleotidase in the muscles of marine vertebrates and invertebrates (total 11 species) were determined. The highest activity of 5'-nucleotidase was found in Blackrock fish Sebastes inermis, which was then used as a material for estimation of subcellular distribution and solubilization of the enzyme. 2. The 5'-nucleotidase of ordinary muscle of the fish Sebastes inermis was found in nuclear, microsomal and cytosolic fractions. About half of the total activity was found in the nuclear fraction, whereas the highest specific activity was observed in the microsomal fraction. 3. Complete solubilization of the enzyme was attained by using a high concentration of detergent such as Triton X-100, CHAPS, octylglucoside, octylthioglucoside and sodium deoxycholate, suggesting that the enzyme was tightly bound to the membrane. 4. Based on the results of solubility and stability tests, Triton X-100 seemed suitable for solubilizing 5'-nucleotidase from the membrane. 5. Microsomal 5'-nucleotidase was an Mg(2+)-activated enzyme, and no inactivation was observed up to 50 mM of Mg2+.     

Subcellular distribution of adenosine system enzymes in the hypothalamus and hippocampus of the rat brain

The subcellular distribution of 5'-nucleotidase and adenosine deaminase in rat brain hypothalamus and hippocampus was studied. In the hippocampus the 5'-nucleotidase activity was shown to be much higher than in the hypothalamus, while the adenosine deaminase activity, contrariwise, is nearly two times as high as that in the hypothalamus. During the analysis of subcellular distribution 5'-nucleotidase and adenosine deaminase were detected in all fractions under study, i. e., in nuclear, soluble, myelin fractions as well as in synaptic membranes, synaptosomes and "pure" mitochondria. The highest 5'-nucleotidase activity was found in the myelinic and synaptic fractions both in the hypothalamus and in the hippocampus. The highest adenosine deaminase activity was detected in the soluble fraction of the above structures. The enzyme activity in synaptic membranes and synaptosomes was nearly two times as low.     

Concanavalin A inhibition of ecto-5'-nucleotidase of intact cultured C6 glioma cells.

A pronounced effect of concanavalin A (Con A) upon activity of ecto-5'-nucleotidase of intact C6 glioma cells in culture has been demonstrated. A near linear rate of decrease in 5'-nucleotidase activity was observed upon treatment with concentrations of Con A up to 0.25 muM. Nonspecific phosphatase activity and Ca2+-dependent ATPase activity were not inhibited by Con A treatment of the cells. Of the total 5'-nucleotidase activity of C6 cells (Vmax = 5.0 mumol of Pi liberated/mg of cell protein/hour), approximately 20% still remained after treatment with high concentrations of Con A. The inhibitory effect of Con A operated to reduce substantially Vmax for ecto-5'-nucleotidase. Inhibition was reversed by briefly incubating the Con A-treated cells with alpha-methyl-D-glucoside, or alpha-methyl-D-mannoside, the later being more effective. These findings suggest that a relatively specific, reversible, inhibition of ecto-5'-nucleotidase results from Con A binding to the surface of the intact cultured mammalian cells.     

Catalytic characteristics of 5'-nucleotidase in the structure of cell membrane rafts in smooth muscles

5'-nucleotidase (EN 3.1.3.5) is widely distributed enzyme occurring in vertebrate, bacterial and plant cells. The main physiological function of 5'-nucleotidase is hydrolysis of 5'-AMP to adenosine and Pi. It was found that the detergent-insoluble membrane domains (rafts) are enriched by proteins possessing high 5'-AMPase activity. This study is aimed to investigate some physical and chemical properties of 5'-nucleotidase, which is present in detergent insoluble membrane domains isolated from pig stomach and lung. It was shown for the first time that catalytic properties of the raft-associated 5'-nucleotidase and of the pure enzyme described in literature differ. Our results demonstrate that the greatest activity of the raft-associated enzyme takes place in the physiological conditions contrary to the pure enzyme. Our data suggest that such changes of 5'-nucleotidase catalytic activity might be due to the disruption of its interaction with membrane rafts.     

Enzymatic activity and in vivo distribution of 5'-nucleotidase, an extracellular matrix binding glycoprotein, during the development of chicken striated muscle.

The ecto-enzyme 5'-nucleotidase isolated from chicken gizzard has previously been shown to be a potent ligand of two glycoproteins of the extracellular matrix, namely fibronectin and laminin. Using immunofluorescent labeling techniques we observed that 5'-nucleotidase codistributed with laminin during the development of chicken striated muscle. In contrast, ecto-5'-nucleotidase was only faintly detectable on cells surrounded by a matrix expressing high levels of fibronectin. This distribution pattern distinguished 5'-nucleotidase from the pluripotent extracellular matrix receptors, chicken beta 1-integrins, which are expressed equally well in muscle and connective tissue. In addition, the specific activity of striated muscle ecto-5'-nucleotidase was stable during development and increased markedly posthatching. At each age considered, this specific activity corresponded to an 80-kDa enzyme which was inhibited by alpha,beta-methyleneadenosine diphosphate or by a monoclonal antibody directed against the smooth muscle isoform of the enzyme. Previous in vitro studies have revealed that 5'-nucleotidase is involved in the spreading of various mesenchyme-derived cells, such as chicken embryonic fibroblasts and myoblasts, on a laminin substrate. A prerequisite to examining a potential in vivo role for 5'-nucleotidase as an extracellular matrix ligand was to study its distribution. In adult muscle, 5'-nucleotidase displayed a more restricted distribution than in embryo. Results show that, in vivo, 5'-nucleotidase is revealed by immunofluorescent labeling using poly- and monoclonal antibodies to chicken gizzard 5'-nucleotidase in two structures, the costameres and myotendinous junctions, which are closely related to the focal adhesion sites observed in cell culture.     

Enzyme-histochemical identification of lymphatic vessels by light and backscattered image scanning electron microscopy

The walls of lymphatics are characterized by strong 5'-nucleotidase activity, whereas those of blood capillaries reveal significantly lower or no activity. Alkaline phosphatase activity, on the other hand, is markedly higher in blood capillaries than in lymphatic vessels. On the basis of such characteristics, lymphatics and blood capillaries were distinguished histochemically in rat stomach using 5'-nucleotidase-alkaline phosphatase double staining. The distribution and intensity of lead-demonstrated 5'-nucleotidase activity in lymphatic vessels could be determined by comparing the images of the same histochemically stained cryostat section as seen by light and backscattered image scanning electron microscopy. The specificity of the 5'-nucleotidase reaction was obtained by inhibiting nonspecific alkaline phosphatase by including L-tetramisole in the 5'-nucleotidase incubation medium. The products of the 5'-nucleotidase activity were deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells.