Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.5 (5'-nucleotidase)
 3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.
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PMID:Plasma membrane localization of alkaline phosphatase in HeLa cells. 5 27

The effects of fixation with various concentrations of glutaraldehyde or formaldehyde, acetone or ethanol, and freeze-drying on 5 phosphatases of Eimeria tenella and chick kidney cell cultures were demonstrated in situ. Gultaraldehyde inactivated the phosphatases more than did the formaldehyde, but the effect of the combination of the 2 (Karnovsky's fixative) was greater than that of either glutaraldehyde or formaldehyde alone. The higher the concentration of aldehyde and the longer the duration of exposure, the greater the inactivation. The order of sensitivity to aldehyde fixation of the enzymes tested was glucose-6-phosphatase greater than thiamine pyrophosphatase greater than 5'-nucleotidase greater than adenosine triphosphatase greater than acid phosphatase. Cytologic detail was preserved more efficiently with glutaraldehyde than with formaldehyde. Optimal preservation of enzyme activity for cytochemistry was with 2% glutaraldehyde for 30 min or 2% formaldehyde for 1 hr for G-6-Pase, TPPase, and 5'-nucleotidase, and with 2% glutaraldehyde or 2% formaldehyde for 2 hr with ATPase and AcPase. Quenching with subsequent fixation in cold acetone or ethanol resulted in complete inactivation of G-6-Pase, TPPase, and 5'-nucleotidase; although cells fixed in this manner yielded large amounts of reaction product for ATPase and AcPase, the distribution was diffuse, and some of it appeared to be artifactual. Quenching with subsequent freeze-drying was unsatisfactory because nearly all of the cell layers rolled off the cover glasses.
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PMID:Effect of fixation on demonstration of phosphatases of Eimeria tenella grown in chick kidney cell cultures. 6 Dec 71

Determinations of protein and phospholipid composition, as well as enzymatic activity, were carried out in plasma membranes isolated from the muscle of rats, after different periods of 20,25-diazacholesterol administration. A decrease in the level of phospholipids, and in the total amount of plasma membrane proteins, connected with a relative reduction in the amount of protein of a molecular weight of 100000 daltons, was found. The activity of (Na+ + K+)-ATP-ase gradually decreased while a reverse tendency was observed in the case of 5'-nucleotidase. Changes in ATP-ase and phospholipids appeared even prior to electrophysiologically recorded signs of the myotonia. The mechanism of these changes and their possible role in myotonia are discussed.
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PMID:Plasma membranes of muscle in experimental myotonia in rats. 6 35

The mean activity of purine 5'-nucleotidase (E.C.3.1.3.5) in lymphocytes was significantly lower in patients with non-familial adult onset "variable" primary hypogammaglobulinaemia than in non-hypogammaglobulinaemic control subjects. These findings suggest that 5'-nucleotidase activity is necessary for normal lymphocyte function. This may be related to its role in facilitating the transfer of newly formed purines across cell membranes by converting them from nucleotides to nucleosides. This enzyme defect may be a characteristic metabolic lesion in at least some of the patients; alternatively, it may be secondary to some other metabolic defect or plasma-membrane change.
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PMID:Lymphocyte purine 5'-nucleotidase edficiency in primary hypogammaglobulinaemia. 6 99

An increase of thymidine kinase [EC 2.7.1.21] activity and decrease of 5'-nucleotidase [EC 3.1.3.5] activity for dTMP were found during hormonal regeneration of the seminal vesicles by daily or single administration of testosterone propionate into mice castrated 2 weeks previously. Actinomycin D injected on day 0 of testosterone treatment completely inhibited both the increase of thymidine kinase and the decrease of 5'-nucleotidase. When injected on day 2, actinomycin D decreased thymidine kinase activity below the control level and 5'nucleotidase activity was not restored to the normal level. The activity of 5'-nucelotidase in a mixed sample, in which seminal vesicles of castrated mice and those of testosterone-treated mice were homogenized together, was intermediate between the activities determined separately. This indicates the absence of any inhibitor of 5'nucleotidase in the regenerating vesicles. Changes in total activity of 5'nucleotidase and total protein content in extracts during various treatments showed that the decrease in specific activity of 5'-nucleotidase in the first 2 days of testosterone treatment was not due to inhibition of enzyme activity but to dilution of the enzyme with other proteins which increased in content more rapidly than 5'-nucleotidase.
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PMID:Changes in enzyme activities of thymidine kinase and 5'-nucleotidase for dTMP during hormonal regeneration of seminal vesicles of mice. 7 66

Large amounts of injected radiolabeled low density lipoproteins have been found by others to accumulate primarily in the liver and studies in various types of isolated cells, including hepatocytes, have indicated the presence of specific cell membrane recognition sites for lipoproteins. In the present studies, the high affinity binding of radiolabeled low density lipoproteins ([125I]LDL, d 1.020--1.063 g/mL) was measured in the major subcellular fractions of porcine liver homogenates. The nuclear and mitochondrial fractions were 1.9- and 1.4-fold enriched in binding activity with respect to unfractionated homogenates and contained 15% and 12% of the total binding activity, respectively. The microsomes, which contained most of the plasma membranes and endoplasmic reticulum, were approximately 4-fold enriched in binding and contained 73% of the binding activity. Microsomal subfractions obtained by differential homogenization and centrifugation procedures were 5.6--7.0-fold enriched in LDL binding and contained 54--58% of the homogenate binding activity. They were separated by discontinuous sucrose density gradient centrifugation into fractions which contained "light" and "heavy" plasma membranes and endoplasmic reticulum. The heavy membrane fraction was 2--4 fold in binding with respect to the parent microsomes (16--22 fold with respect to the homogenate). There was no enrichment of binding activity in the other two fractions. Two plasma membrane "marker" enzymes, nucleotide pyrophosphatase and 5'-nucleotidase, were also followed. Of the two, binding in the sucrose density gradient subfractions most closely followed nucleotide pyrophosphatase, which was also most highly enriched (3.2--3.3-fold) in the heavy membrane fraction, but did not follow it exactly. The enzyme was 2-fold richer in the light membranes than in the parent microsomes, though the light membrane binding activity was only 0.4--1.4 times that of the parent microsomes. High affinity binding was time and temperature dependent, saturable, and inhibited by unlabeled low density lipoproteins but not by unrelated proteins. Binding was stimulated 2--3 fold Ca2+, was not affected by treatment with Pronase or trypsin and was inhibited by low concentrations of phospholipids and high density lipoproteins (HDL). Heparin-Mn2+ treatment of HDL did not affect its ability to inhibit [125I] LDL binding. The LDL recognition site was distinct from the liver membrane asialoglycoprotein receptor; LDL binding was not inhibited by desialidated fetuin. We conclude that porcine liver contains a high affinity binding site that recognizes features common to both pig low density and high density lipoproteins. Further studies may elucidate the significance of this binding site in lipoprotein metabolism.
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PMID:Isolation of a porcine liver plasma membrane fraction that binds low density lipoproteins. 8 56

Extracellular membraneous vesicles of GRSL leukaemia cells were isolated from the ascites fluid bathing the cells in vivo, and from cell washes. Mammary tumour virus-induced antigens (MLr) expressed on the surface of the cells are enriched on these vesicles as compared to plasma membranes isolated from the cell homogenate. The lipid fluidity of the vesicles is much smaller than that of the plasma membranes, and the content of the pertinent lipid parameters, cholesterol and sphingomyelin, are accordingly greatly increased. The extracellular vesicles which are also enriched in sialic acid and 5'-nucleotidase are apparently derived from the plasma membrane, probably at least partly by exfoliation of selected parts or domains of the surface of living cells. An analogy between this shedding of vesicles, the formation of endocytotic vesicles and the budding of viruses is noted; all these processes select or assemble rigid lipid domains of the cell membrane. The possible participation of surface microvilli and sub-lethal autolysis in the process of shedding is discussed.
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PMID:Rigid plasma-membrane-derived vesicles, enriched in tumour-associated surface antigens (MLr), occurring in the ascites fluid of a murine leukaemia (GRSL). 8 6

The use of enzymes as markers of T or B cells in tissue sections has been studied in mouse lymphoid tissue and lymph nodes from the gerbil, rat and cat. Lymphocytes in the T-cell areas of murine lymph nodes and spleen contained discrete dots of non-specific esterase and N-acetyl-beta-D-glucosaminidase (beta-glucosaminidase) activity, with weak acid phosphatase activity. Lymphocytes in the B-cell areas lacked this discrete staining. Cortical thymocytes contained slight esterase activity while medullary thymocytes were strongly positive for both esterase and beta-glucosaminidase. Lymphocytes with a T-cell staining pattern were only occasionally seen in lymph nodes from Nude (nu/nu) mice. ATPase staining was restricted to lymphocytes in the B-cell areas; weak 5'-nucleotidase staining was only present in a frew lymphocytes in both T- and B-cell areas. Blast cells stimulated by in vivo injection of ConA or PHA in the mouse showed strong discrete enzyme activity for non-specific esterase and beta-glucosaminidase. Lipopolysaccharide-stimulated blast cells and cells within germinal centres lacked this discrete staining. Comparison of lymph nodes from the gerbil, rat and cat suggested at least on enzymes as a T-cell marker in each species although considerable variation in staining profiles was seen in the different species.
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PMID:Cytochemical identification of T and B cells in situ in mouse lymphoid tissue and lymph nodes from the rat, gerbil and cat. 8 10

Human pancreatic exocrine adenocarcinoma cells established in tissue culture expressed both HLA and beta 2-microglobulin (beta 2M). Plasma-membrane components of this pancreatic cancer cell line were purified from plasma membrane fractions enriched by sucrose density-gradient centrifugation, using immunoaffinity chromatography on immobilized anti-human beta 2M antibody. Both rabbit and mouse monoclonal anti-beta 2M IgG were used, with a 20--25-fold overall purification of 5'-nucleotidase. The method was applicable to 5 x 10(7) cells and permitted the solubilization of membranes retained on the column, with the selective desorption of components not associated with beta 2M before the subsequent elution at pH 3 of beta 2M-associated macromolecules. The acid eluate contained one major and two minor bands in the 40--45,000 mol.-wt range with two additional enriched components of 18,000 and 22,000 dalton. A major carbohydrate-containing component of high mol. wt was also found to be associated with the pancreatic cancer-cell plasma membrane.
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PMID:Isolation of plasma-membrane components from cultured human pancreatic cancer cells by immuno-affinity chromatography of anti-beta 2M sepharose 6MB. 9 30

Plasma membranes from normal, full-term human placental trophoblast have been isolated by a new procedure. The method depends upon isopycnic zonal centrifugation using linear sucrose/Ficoll density gradients. Enrichment of plasma membrane marker enzymes with respect to trophoblast homogenate is found in two distinct peaks (designated B and D) of the fractionated effluent recovered from the rotor. Fraction B is enriched with membrane-bound alkaline phosphatase and 5'-nucleotidase, but not with (Na+, K+)-ATPase of F(-)-stimulated adenylate cyclase. It is suggested that this material is derived from the maternal-facing microvillous plasma membrane. Fraction D, enriched with (Na+, K+)-ATPase, F(-)-stimulated adenylate cyclase and, to a smaller extent, with 5'-nucleotidase and alkaline phosphatase is, by exclusion, proposed to be derived from the fetal-facing basal plasma membrane. Both plasma membrane fractions are shown to be free of appreciable contamination, using specific markers for endoplasmic reticulum, mitochondria, nuclei and lysosomes. The separation of the two membrane fractions is shown to depend both upon these membranes forming closed vesicles during homogenization and upon the buoyant densities of such vesicles differing in such a way that microvillous plasma membranes band at a lower density than basal plasma membranes. No separation of the membranes is achieved in gradients in which the vesicles are collapsed.
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PMID:Separation of the microvillous (maternal) from the basal (fetal) plasma membrane of human term placenta: methods and physiological significance of marker enzyme distribution. 9 48


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