Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 specificity of lactoperoxidase-catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques. Light microscope autoradiography of sectioned cells shows the incorporated label to be localized primarily at the periphery of the cell. Most of this label can be released from the cell by trypsin but not by collagenase or hyaluronidase. The label is recovered from the cells as either monoiodotyrosine or diiodotyrosine after hydrolysis of cell extracts with a mixture of proteolytic enzymes. The label co-purifies during cell fractionation with an authentic liver cell plasma membrane marker enzyme, 5'-nucleotidase. Thus, the incorporated iodide is itself a valid marker for those membrane polypeptides having tyrosine residues accessible to the lactoperoxidase. The polypeptide complexity of the purified plasma membrane was examined by high resolution dodecyl sulfate-polyacrylamide gel electrophoresis. At least 50 polypeptides in the membrane are accessible to iodination. These polypeptides probably represent the bulk of the protein mass of the membrane and iodinating them does not affect cell viability, growth rate, or cell function. Labeling experiments with fucose and glucosamine show that at least nine of the iodinated peptides may be glycoproteins.
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PMID:Proteins of the hepatoma tissue culture cell plasma membrane. 0 57

NIL 8 hamster fibroblast cells were labeled by lactoperoxidase-catalyzed iodination. Their membranes were fractionated by sedimentation-rate and isopycnic zonal centrifugation. All the iodinated proteins except the very prominently labeled high molecular weight protein (greater than 200,000 daltons) were located in a fraction identified enzymically and compositionally as plasma membrane. The high molecular weight protein that was previously shown to be sensitive to virus transformation (Hynes, 1973) is concentrated in a very high density particle (rho equals 1.253-1.259) which contains mainly carbohydrate and protein and very low levels of lipid. 5'-nucleotidase was the only enzyme reproducibly demonstrated in this fraction, and electron micrographs revealed a predominantly amorphous morphology together with a few membraneous structures. The iodine label in this fraction was very sensitive to trypsinization prior to homogenization. All the available evidence indicates that this fraction is derived from the surface coat. Mitochondria, nuclei, and soluble protein were labeled to an insignificant extent. The presence of the iodinated surface proteins associated with the endoplasmic reticulum fraction is discussed in the light of these results.
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PMID:The location of proteins labeled by the 125I-lactoperoxidase system in the NIL 8 hamster fibroblast. 12 85

Hepatocytes from rats were isolated by treatment with trypsin and cultured. Plasma membranes at different culture stages were observed by electron microscopy. The activities of 5' nucleotidase and adenosinetriphosphatase on the plasma membranes were examined. The cell coat was also studied by use of the concanavalin A-peroxidase technique. The surfaces of single cells, covered with microvilli, are the site of adenosinetriphosphatase activity only and are devoid of 5'-nucleotidase activity. After a few h of culture, the cells are grouped together in tight clusters or long trails and are separated by an intercellular space of 250 A, partially permeable to lanthanum nitrate. The juxtaposed plasma membranes on which 5'-nucleotidase and adenosinetriphosphatase activities occur also delimit spaces similar to bile canaliculi. The formation of junction complexes and their permeability to lanthanum nitrate was also studied. No enzymatic activity is observed at the junctions. The numerous tight junctions, impervious to the tracer, are always accompanied by a profusion of microfilaments. Mature desmosomes are rare, and are present only in the form of "maculae adhaerentes diminutae." The gap junctions, nearly always permeable to the tracer, form rapidly and assume a variety of shapes (trail, bulge and ring-like), the significance of which is open to discussion. The use of concanavalin A permits localization of the free sugar sites on the surface of the cells, in the pinocytotic vesicles and in the internal space of the gap junctions.
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PMID:Differentiation of the plasma membrane of hepatic cells in monolayer cultures. 13 45

1. Isolated mouse spleen lymphocytes hydrolysed UDP-galactose added to the medium. Nucleotide pyrophosphatase activity that accounted for this hydrolysis was enriched to a similar extent as alkaline phosphodiesterase and 5'-nucleotidase in a lymphocyte plasma-membrane fraction. 2. The cell surfaces of mouse spleen and thymus lymphocytes were iodinated with 125I by using the lactoperoxidase-catalysis method. Detergent extracts of the cells were mixed with a purified anti-(mouse liver plasma-membrane nucleotide pyrophosphatase) antiserum and the immunoprecipitates analysed by polyacrylamide-gel electrophoresis. Only one major radioactive component, similar in size (apparent mol.wt 110000-130000) to the liver enzyme, was observed. 3. Electrophoresis of an iodinated spleen plasma-membrane fraction indicated peaks of radioactivity, including one of apparent mol.wt 110000-130000. 4. When detergent extracts of spleen lymphocytes were passed through a Sepharose-bead column containing covalently attached anti-(nucleotide pyrophosphatase) antiserum, the nucleotide pyrophosphatase activity was retained by the beads, whereas protein and leucine naphthylamidase activity were eluted. 5. The results indicate that nucleotide pyrophosphatase and alkaline phosphodiesterase activities are due to the location of the same or similar enzymes at the outer aspect of the lymphocyte plasma membrane. Some possible functions of enzymes at this location are discussed.
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PMID:Location of nucleotide pyrophosphatase and alkaline phosphodiesterase activities on the lymphocyte surface membrane. 18 74

Activities of a broad spectrum of enzymes were studied histochemically in renal adenocarcinomas induced in young male F344 rats by chronic dietary administration of the carcinogen N(4'-fluoro-4-biphenylyl)acetamide. Enzymes included were: dehydrogenases of glucose-6-phosphate, lactate, succinate, malate, and alpha-glycerophosphate; peroxidase (catalase); glucose-6-phosphatase; alkaline and acid phosphatase; Mg2+ ATPase; 5'-nucleotidase; and aminopeptidase. Levels of enzyme activity were estimated visually and scored from 0 (not detectable) to a maximum of 5 (intense). Comparison of estimated activity for each enzyme was made between small neoplastic nodules (stage III tumors) and large adenocarcinomas (stage IV tumors) and between tumors and portions of normal proximal tubules in parenchyma of kidneys from untreated control rats. The results, which revealed nearly identical levels of activity for most enzymes in both stages III and IV tumors, suggested similar metabolic and biologic behavior of these lesions. However, when data for tumors were compared with data for normal proximal tubules, striking differences were observed consistent with: 1) a marked shift of energy metabolism from oxidative to glycolytic production of ATP, with a corresponding reduction in mitochondrial respiration; and 2) simplification of plasma membrane specializations that were possibly associated with a reduction or loss of transport function. These findings were compared with other histochemical, biochemical, and ultrastructural studies of renal adenocarcinomas in rats and man.
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PMID:Adenocarcinoma of the kidney. II. Enzyme histochemistry of renal adenocarcinomas induced in rats by N-(4'-fluoro-4-biphenylyl)acetamide. 18 77

Ovine prolactin, labelled with 125I by either lactoperoxidase or a mild chloramine T method, bound to receptors from the pigeon crop sac mucosa cells of prolactin-injected pigeons. Binding was demonstrated in a crude homogenate of mucosal cells removed from the crop by scraping and in a subcellular fraction in which 5'-nucleotidase activity was enhanced two- to threefold. The binding was specific, dependent on time, temperature and the concentration of receptors and had a dissociation constant of 7 X 10(-10) mol/l. The binding capacity of the crop tissue was 71 fmol/mg membrane protein. Nine purified preparations of prolactin from four species were assayed by local pigeon crop sac bioassay and by radioreceptor assay. The two methods were highly correlated (r = 0.934). The regression equation was radioreceptor assay = 1.22 bioassay--0.18 indicating a 1 : 1 correspondence between the two methods for prolactin purified from sheep, rat, horse and pig anterior pituitary glands.
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PMID:A pigeon crop sac radioreceptor assay for prolactin. 21 43

Monocytes of healthy, full-term newborns were isolated from cord blood, and functional and biochemical activities were quantitated. The yield of monocytes per milliliter of cord blood was 60% greater than that from the peripheral blood of healthy adults. Placental monocytes were initially less well spread than cells from adults, but no other morphological differences were noted. During 4 days of in vitro cultivation, placental monocytes secreted lysozyme at a constant rate, lost peroxidase activity, and increased 5'-nucleotidase activity 15- to 25-fold. Similar findings were obtained with monocytes from adults. Placental monocytes also displayed Fc and complement receptor activity. Ingestion and intracellular multiplication of Toxoplasma gondii were identical in normal placental and adult monocytes. Furthermore, toxoplasma multiplication was significantly inhibited by cells from both sources when the monocytes were preincubated with supernatants prepared from sensitized lymphocytes and toxoplasma antigen. Monocytes from newborns were competent cells in terms of the specific functions and activities we examined.
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PMID:Monocyte function in human neonates. 64 Jul 36

Two established techniques for analysis of plasma membranes, namely, lactoperoxidase catalyzed surface radioiodination of intact cells and bulk membrane isolation following disruption of cells by shear forces, were applied in studies of membrane proteins of continuously cultured cells of the monoclonal T lymphoma line WEHI-22. It was found that macromolecular 125I-iodide incorporated into plasma membrane proteins of intact cells was at least as good a marker for the plasma as was the commonly used enzyme 5'-nucleotidase. T lymphoma plasma membrane proteins were complex when analysed by polyacrylamide gel electrophoresis in sodium dodecylsulphate-containing buffers and more than thirty distinct components were resolved. More than fifteen of the components observed on a mass basis were also labelled with 125I-iodide. Certain bands, however, exhibited a degree of label disproportionate to their staining properties with Coomassie Blue. This was interpreted in terms of their accessibility to the solvent in the intact cells.
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PMID:Plasma membrane of a murine T cell lymphoma: surface labelling, membrane isolation, separation of membrane proteins and distribution of surface label amongst these proteins. 108 76

1. Human tumour KB cells growing in suspension culture were labelled by lactoperoxidase-catalysed iodination. Several major radioactively labelled proteins were detected by poly-acrylamide-gel electrophoresis in sodium dodecyl sulphate. 2. After reduction with 2-mercaptoethanol the major radioactive electrophoretic bands migrated as substances with apparent molecular weights of about 90,000, 70,000, 60,000, 50,000 and 34,000 and corresponded closely to the positions at which the major glycosylated polypeptide subunits of KB-cell homogenates migrated during electrophoresis under the same conditions. 3. All the iodinated protein bands except one were present in purified preparations of KB plasma membranes. 4. Most of the 50,000-molecular-weight species, supposedly a surface protein component labelled during iodination of intact and viable KB cells by a non-penetrating enzyme reagent, appeared in a crude nuclear pellet during fractionation. 5. The glyco-protein nature of the major external iodinated species of KB cells was confirmed by adsorption chromatography of these substances, dissolved in low concentrations of Triton X-100, on a lectin-Sepharose column. Two major enzyme markers of the KB plasma membrane, 5'-nucleotidase and alkaline phosphatase were also found to be glycoproteins. 6. Enzyme-catalysed incorporation of radioactive iodine into a fraction of low molecular weight and soluble in chloroform-methanol mixtures also occurred during lactoperoxidase treatment of intact KB cells. The partial characterization of this fraction is briefly described.
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PMID:Surface labelling for human tumour KB cells. Iodination and fractionation of membrane glycoproteins. 120 Oct 9

Plasma membranes were prepared from the human lymphocyte cell line WIL23A by hypotonic swelling, Dounce homogenization, differential and equilibrium centrifugation. The resulting vesiculated membrane fragments were found to have densities of 1.10 and 1.17 g/ml, and were defined by lactoperoxidase mediated whole cell iodination, L-[3H] fucose incorporation, 5'-nucleotidase activity (EC 3.1.3.5) and electron micrographic visualization. Recovery of plasma membrane from whole cell homogenates was estimated to be approximately 30-35% as judged by the recovery of 125I-labeled cell surface protein. When plasma membranes were prepared from cells which had been incubated for 18 h in the presence of 0.5 muCi/ml [3H] thymidine such that greater than 10(9) acid insoluble counts could be demonstrated in the whole cell homogenates, no [3H] thymidine label and presumably, therefore, no DNA, could be shown to be coincident with either the 1.10 or 1.17 density. Similar experiments with [3H] uridine suggested that 90% of the plasma membranes did not contain RNA, while 10% remained questionable.
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PMID:Investigation of human lymphocyte plasma membrane associated nucleic acid. 126 57


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