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)

1. Polypeptides of liver plasma membrane fractions enriched in three surface domains of hepatocytes, blood-sinusoidal, lateral and bile canalicular, were analysed by isoelectric focusing (IEF) and non-equilibrium pH gel electrophoresis (NEPHGE) across a wide pH range, followed by SDS/PAGE. The overall Coomassie Blue-stained polypeptide patterns in the fractions were different. lateral plasma membrane fractions contained a characteristically higher number of polypeptides focusing at the basic pH range, whereas few basic polypeptides were present in sinusoidal plasma membrane fractions. The glycoproteins in these plasma membrane fractions stained by a lectin overlay technique with radio-iodinated concanavalin A, wheat-germ agglutinin and a slug lectin, were also different. 2. The polypeptides and glycoproteins of 'early' and 'late' endosome fractions were also compared by two-dimensional electrophoresis. Their composition was shown by Coomassie Blue staining, lectin overlay staining and in membranes metabolically labelled with [35S]methionine to be generally similar. The glycoproteins of sinusoidal plasma membranes and early and late endosomes were generally similar, but major differences in polypeptides of molecular mass 20-50 kDa, pI 7.5-8.5, in plasma membranes and endosomes were demonstrated, with a specific population of basic (pI 8-9) low-molecular-mass polypeptides being present at highest levels in 'late' endosomal fractions (shown by Coomassie Blue staining). 3. Analysis of the distribution of three specific membrane glycoproteins identified by using immunoblotting techniques showed that the asialoglycoprotein and the divalent-cation-sensitive mannose 6-phosphate receptors were present in sinusoidal plasma membrane and in early and late endocytic fractions: they were not detected in canalicular plasma membrane fractions. In contrast, 5'-nucleotidase was detected in all fractions examined. The role of the endocytic compartment in regulating trafficking pathways between the plasma membrane domains of the hepatocyte is discussed.
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PMID:A two-dimensional electrophoretic analysis of the proteins and glycoproteins of liver plasma membrane domains and endosomes. Implications for endocytosis and transcytosis. 217 96

1. Liver plasma membranes originating from the sinusoidal, lateral and canalicular surface domains of hepatocytes were covalently labelled with sulpho-N-hydroxysuccinamide-biotin. After solubilization in Triton X-114, treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), two-phase partitioning and 125I-streptavidin labelling of the proteins resolved by PAGE, six major polypeptides (molecular masses 110, 85, 70, 55, 38 and 35 kDa) were shown to be anchored in bile canalicular membrane vesicles by a glycosyl-phosphatidylinositol (G-PI) 'tail'. 2. Permeabilized 'early' and 'late' endocytic vesicles isolated from liver were also examined. Two polypeptides (110 and 35 kDa) were shown to be anchored by a G-PI tail in 'late' endocytic vesicles. 3. Analysis of marker enzymes in bile-canalicular vesicles treated with PI-PLC showed that 5'-nucleotidase and alkaline phosphatase, but not leucine aminopeptidase and ecto-Ca2(+)-ATPase activities were released from the membrane. A low release and recovery of alkaline phosphodiesterase activity was noted. The cleavage from the membrane of 5'-nucleotidase as a 70 kDa polypeptide was confirmed by Western blotting using an antibody to this enzyme. 4. Antibodies raised to proteins released from bile-canalicular vesicles by PI-PLC treatment, and purified by partitioning in aqueous and Triton X-114 phases, localized to the bile canaliculi in thin liver sections. Antibodies to proteins not hydrolysed by this treatment stained by immunofluorescence the sinusoidal and canalicular surface regions of hepatocytes. 5. Antibodies generated to proteins cleaved by PI-PLC treatment of canalicular vesicles were shown to identify, by Western blotting, a major 110 kDa polypeptide in these vesicles. Two polypeptides (55 and 38 kDa) were detected in MDCK and HepG-2 cultured cells. 6. Since two of the six G-PI-anchored proteins targeted to the bile-canalicular plasma membrane were also detected in 'late' endocytic vesicles, the results suggest that a junction where exocytic and endocytic traffic routes meet occurs in a 'late' endocytic compartment.
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PMID:Priority targeting of glycosyl-phosphatidylinositol-anchored proteins to the bile-canalicular (apical) plasma membrane of hepatocytes. Involvement of 'late' endosomes. 217 97

Rat liver 5'-nucleotidase was purified from a crude microsomal fraction, and its molecular mass was estimated to be 73 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein was subjected to cleavage with CNBr or lysyl endopeptidase, and the resulting 21 peptides as well as the NH2 terminus of the native protein were sequenced by Edman degradation. For further information on the molecular structure, we constructed a lambda gt11 liver cDNA library and isolated two cDNA clones for 5'-nucleotidase, lambda cNTP6 and lambda cNT34. The 3.2-kilobase cDNA insert of lambda cNTP6 contains an open reading frame that encodes a 576-residue polypeptide with a calculated size of 63,965 Da, which is in reasonable agreement with that of 5'-nucleotidase (62 kDa) immunoprecipitated from cell-free translation products. The NH2-terminal 28 residues comprise a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. The predicted structure contains all the other peptide sequences determined by Edman degradation. Five potential N-linked glycosylation sites are found in the molecule, accounting for the difference in mass between the precursor and mature forms. Another characteristic feature is that the primary structure contains a highly hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. In fact, labeling experiments of rat hepatocytes demonstrated that 3H-labeled compounds such as ethanolamine, myo-inositol, and palmitic acid, components of the glycolipid anchor, were incorporated into 5'-nucleotidase. Phosphatidylinositol-specific phospholipase C released 5'-nucleotidase from the cell surface, and the released protein no longer contained the radioactivity of [3H]palmitic acid incorporated.
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PMID:Primary structure of rat liver 5'-nucleotidase deduced from the cDNA. Presence of the COOH-terminal hydrophobic domain for possible post-translational modification by glycophospholipid. 229 43

Epidermal growth factor (EGF) is now well known as a potent mitogen and differentiation factor for a variety of cells both in vivo and in vitro. Like other polypeptide hormones, EGF initially binds to a specific plasma membrane receptor on the target cells. In this study, we investigated the effect of streptozotocin-induced diabetes on EGF receptors on rat liver plasma membranes. An apparent increase in serum glucose concentration was observed in diabetic rats, and treatment of diabetic animals with insulin normalized the glucose concentration to the control level. There was no marked difference in hepatic membrane markers among the control, diabetic and insulin-treated diabetic animals, as judged by protein, sialic acid contents, and phosphodiesterase I and 5'-nucleotidase activities. The binding of 125I-EGF to membranes was found to be significantly lower in diabetic than in control animals. The value in diabetic animals was about 55% of the control level. Insulin treatment of diabetic animals restored the binding of 125I-EGF to the control level, whereas triiodothyronine (T3) treatment had no effect. Scatchard analysis of the binding data clearly showed that the decrease in EGF binding was due to a decrease in the number of receptors rather than to a change in receptor affinity. The decrease in EGF receptor number in diabetic animals was also confirmed by an experiment on affinity labeling of EGF receptors. EGF stimulated the phosphorylation of hepatic EGF receptors (molecular weight = 170,000). The rates of basal and EGF-stimulated phosphorylation of the receptors were lower in diabetic than in control animals. Insulin treatment of diabetic animals restored the phosphorylation activity to control level, whereas T3 treatment had no apparent effect. There was no significant difference in serum EGF concentration among the control, diabetic and insulin-treated diabetic animals. These results indicate that insulin deficiency in vivo causes a decrease in hepatic EGF receptor number, and suggest that the actions of EGF on hepatocytes may also be affected by diabetes mellitus since the effects of EGF are receptor-mediated.
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PMID:[Effect of experimental diabetes on epidermal growth factor (EGF) receptors in the rat liver]. 253 89

Biochemical information about receptors for adrenergic and opioid neurotransmission in submucosal plexus (SMP) is unavailable. We have purified a fraction P2 enriched in synaptosomes and neuronal membranes (high [3H]saxitoxin binding and high vasoactive intestinal polypeptide immunoreactivity, low activity of 5'-nucleotidase) from the canine small intestine SMP. The synaptosomal fraction (fraction P2) also contained a high density of opioid diprenorphine binding sites of high affinity. [3H]rauwolscine binding was enriched both in fraction P2 and in a microsomal fraction. Competition experiments using several adrenergic and opioid receptor ligands revealed that opioid receptors were approximately 64% mu-, 24% delta-, and 12% kappa-subtypes and that adrenoceptors on fraction P2 were alpha 2-subtype but that there was a heterogeneous population of alpha 2-adrenoceptors. These studies show that a fraction enriched in synaptosomes and neural membranes from the canine intestine SMP contains opioid as well as alpha 2-adrenoceptors, that all three subtypes of opioid receptors seem to be present with mu-receptors predominant, and that subtypes of alpha 2-adrenoceptors appear to be present.
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PMID:Biochemical studies on opioid and alpha 2-adrenergic receptors in canine submucosal neurons. 254 1

Membrane-bound 5'-nucleotidase from Vibrio parahaemolyticus was solubilized and purified using a nonionic detergent, heptyl-beta-D-thioglucoside, and was characterized. This enzyme required Mg2+ for activity, maximum activity being observed at 5 and 20 mM Mg2+ with AMP and ATP, respectively, as substrates. Of the divalent cations tested, Mn2+ and Co2+ were able to replace Mg2+ partially, whereas Ca2+ was ineffective. Zinc strongly inhibited the enzyme activity and Ni2+ caused partial inhibition. This enzyme required Cl- for activity, the optimal concentration being 20 mM or more. The order of effectiveness of anions was Cl- greater than Br- greater than I- approximately NO3-. Sulfate and acetate were ineffective. The optimal pH was 8.0. The activity of the purified enzyme was stimulated by the addition of lipid to the assay mixture. This enzyme hydrolyzed all 5'-nucleotides tested, but did not hydrolyze 3'-nucleotides or ribose 5-phosphate. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appeared to be a single polypeptide, with a molecular weight of 72 kDa.
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PMID:Purification and characterization of membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus. 254 26

Bungarus candidus venom exhibited high hyaluronidase, acetylcholinesterase and phospholipase A activities; low proteinase, 5'-nucleotidase, alkaline phosphomonoesterase and phosphodiesterase activities and moderately high L-amino acid oxidase activity. SP-Sephadex C-50 ion exchange chromatographic fractionation of the venom and Sephadex G-50 chromatography of the major lethal venom fractions indicate that the venom contains at least two highly lethal, basic phospholipases A with LD50 (i.v.) values of 0.02 micrograms/g (F6A) and 0.18 micrograms/g (F4A), respectively; as well as two polypeptide toxins with LD50 (i.v.) values of 0.17 micrograms/g and 0.83 micrograms/g, respectively. The major lethal toxin is the basic lethal phospholipase A, F6A, which accounts for approximately 13% of the venom protein and has a mol. wt of 21,000.
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PMID:The lethal and biochemical properties of Bungarus candidus (Malayan krait) venom and venom fractions. 279 37

To investigate the mechanism by which glucocorticoids inhibit glucose transport in peripheral tissues, we have used a monoclonal antibody directed against the human glucose transporter to measure the relative amounts of glucose transporter polypeptide in various cell fractions of human foreskin fibroblasts after treatment with and without dexamethasone. In cells treated for 4 h with 100 nM dexamethasone, a decrease of 48% in glucose transport was accompanied by a decrease of 40% in the amount of glucose transporter polypeptide in a plasma membrane fraction enriched 10-fold in 5'-nucleotidase activity and a 78% increase in the amount of transporter polypeptide in a fraction of putative intracellular membranes, designated P2. There was no significant change in the amount of transporter polypeptide in whole cell lysates. Insulin (200 nM) stimulated glucose transport in basal fibroblasts by only 9%. However, addition of insulin for 30 min to cells that had been treated for 4 h with dexamethasone completely reversed the dexamethasone-induced decrease in glucose transport and also reversed the dexamethasone-induced changes in glucose transporter polypeptide content of the plasma membrane and P2 fractions. From these observations we conclude that dexamethasone decreases glucose transport by causing translocation of glucose transporters from the plasma membrane to an internal location and that insulin reverses the dexamethasone effect by reversing the translocation.
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PMID:Dexamethasone causes translocation of glucose transporters from the plasma membrane to an intracellular site in human fibroblasts. 282 29

Primary cultures of Schwann cells were labeled by indirect immunofluorescence using an antibody directed against 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Schwann cells which had been maintained in culture for 8 weeks were labeled with this antibody. Immunoblot analysis of Schwann cell homogenates revealed a single band with a molecular weight of 54,000 daltons which corresponded to a single immunoreactive polypeptide present in myelin prepared from rat sciatic nerve. The subcellular localization of CNPase was examined by fractionation of cultured Schwann cell homogenates with linear sucrose gradients. The distribution of CNPase paralleled that of 5'-nucleotidase, a putative marker for plasma membranes. These results suggest that CNPase is localized on the plasma membranes of Schwann cells and is expressed by the cells in the absence of an axonal stimulus.
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PMID:Localization of 2',3'-cyclic nucleotide 3'-phosphodiesterase on cultured Schwann cells. 298 27

5'-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5'-nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E.M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J.P. (1984) Biochem. J. 221, 369-377). Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5'-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5'-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5'-nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver.
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PMID:An improved procedure for purifying 5'-nucleotidase from various sources. Evidence for tissue and species differences in their molecular mass and affinity for F-actin. 299 65


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