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)

Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).
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PMID:Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium. 616 62

5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) of bovine milk fat globules can be solubilized by deoxycholate from either isolated globule membranes or washed cream. The solubilized and membrane-bound enzymes exhibit similar Km values and are inhibited by concanavalin A by an apparent noncompetitive process. The soluble enzyme shows positive cooperativity for the inhibition (Hill coefficient of 2) at 37 degrees C, but the membrane enzyme exhibits essentially no cooperation effect. At lower temperatures (5 or 20 degrees C) the cooperative effect in the inhibition of the soluble enzyme is lost. Colchicine and cytochalasin D failed to induce cooperativity of the concanavalin A inhibition of the membrane enzyme, but induction cooperativity occurred when membranes were extracted with glycine/EDTA/mercaptoethanol, releasing a major protein component with a polypeptide molecular weight of 155 000. We suggest that the interaction of this component with the membrane imposes restraints on the behavior of the nucleotidase which are reflected in the cooperativity of the inhibition of the enzyme by concanavalin A.
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PMID:Cooperativity of the concanavalin A inhibition of bovine milk fat globule membrane 5'-nucleotidase. Response to extraction of nucleotidase and of putative cytoplasmic surface coat components. 624 89

Synaptosomes were prepared from rat cortex by subjecting a washed crude mitochondrial pellet to centrifugation first on discontinuous Ficoll-isotonic sucrose gradients and then on discontinuous sucrose gradients. The synaptosome fraction, collected from the 7.5-14% Ficoll band (II), was further separated into two additional fractions, designated IIA and IIB, which bank at the 0.32-1.05 M and at the 1.05-1.6 M sucrose interfaces, respectively. Electron microscopic analysis showed that fraction IIB contained synaptosomes and extra terminal mitochondria and was essentially free of membrane fragments. Further characterization showed that IIB contained 69% of the protein and 83% of the lactic dehydrogenase activity of fraction II and had a specific activity of a 2',3'-cyclic nucleotide 3'-phosphohydrolase approximately 1% of that obtained with myelin. Fraction IIA had approximately 50% the specific activity of the 2',3'-cyclic nucleotide 3'-phosphohydrolase found in myelin. Synaptic plasma membranes were prepared by lysing fraction IIB in 1 mM sodium phosphate, 0.1 mM EDTA at pH 8.5 and subjecting this preparation to centrifugation on a discontinuous sucrose density gradient. Enzymatic analysis indicated that membranes banding at the 0.6-0.8 M sucrose interface had high specific activities of plasma membrane enzymes (e.g. acetylcholinesterase, ATPase, 5'-nucleotidase). The specific activity of the (Na+ + K+)-ATPase in the purified membrane preparation was 8-fold higher than that in the original homogenate. Specific activities of various marker enzymes indicated that the composition of these membrane preparations for the most part was synaptic plasma membranes, approximately 7% mitochondrial outer membranes and 3% a membrane containing 2',3'-cyclic nucleotide 3'-phosphohydrolase activity. The polypeptide compositions of three possible contaminating membranes and of synaptic membranes were compared by electrophoresis in 6-20% gradient polyacrylamide gels in the presence of sodium dodecyl sulfate. Whereas mitochondrial and myelin membranes had distinct compositions, the compositions of the microsomal and synaptosomal plasma membranes were similar. Synaptic plasma membranes contained at least 27 polypeptides; the three major polypeptides had molecular weights of 103,000; 54,000; and 50,000. The major polypeptides of soluble synaptosomal proteins had molecular weights of 54,000 and 42,000.
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PMID:An improved method of preparing rat brain synaptic membranes. Elimination of a contaminating membrane containing 2',3'-cyclic nucleotide 3'-phosphohydrolase activity. 624 53

High voltage free flow electrophoresis has been applied to the separation of human platelet membranes. After short treatment with neuraminidase at the whole cell level, three membrane vesicle subpopulations have been isolated. Using a surface label (125I-labeled Lens culinaris lectin), the marker enzyme NADH-cytochrome c reductase, and lipid analysis, two of the fractions have been identified as of surface origin and the other consists of intracellular membrane elements. The distribution of adenylate cyclase, leucyl aminopeptidase, 5'-nucleotidase and Ca2+-ATPase has also been investigated, and their usefulness as markers for the different membrane fractions has been evaluated. All three fractions are vesicular but differ in size and character. Their phospholipid and cholesterol contents have been determined, and the cholesterol/phospholipid ratios of the two surface fractions are over twice that of the intracellular membrane, which also has a significantly lower microviscosity as determined by fluorescence polarization using diphenyl hexatriene. The polypeptide profiles from sodium dodecyl sulfate-polyacrylamide gel electrophoresis are particularly distinctive, with actin present in the two surface membrane fractions and absent from the intracellular membranes. Myosin, confirmed by its ATPase characteristics, is almost exclusively localized in one of the surface membrane fractions, and actin-binding protein is a prominent feature of the other.
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PMID:Characterization of human platelet surface and intracellular membranes isolated by free flow electrophoresis. 626 Jul 85

1. A variety of detergents were used to solubilize 5'-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5'-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.
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PMID:Solubilization and purification of rat liver 5'-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent. 628 94

By means of DEAE-Sephadex A-50 column chromatography and gel filtrations on Sephadex G-75, Sephacryl S-300 and Sephadex G-100, successively, a potent 5'-nucleotidase was purified from Trimeresurus gramineus venom. The venom 5'-nucleotidase is a single polypeptide chain and homogeneous as judged by SDS-polyacrylamide gel electrophoresis. It is a thermostable glycoprotein consisting of 589 amino acid residues. Its molecular weight was estimated to be 74,000 by SDS-polyacrylamide gel electrophoresis. It possessed nucleotidase activities toward adenosine monophosphate and adenosine diphosphate. The specific activities toward AMP and ADP were 504 +/- 28 and 101 +/- 8 micrograms Pi/min per mg, respectively. Pre-incubation of this venom's 5'-nucleotidase with ADP resulted in the cleavage of ADP and formation of adenosine. The 5'-nucleotidase activity was inhibited by EDTA. Both Zn2+ and Co2+/- reversed the inhibitory effect of EDTA. In rabbit platelet-rich plasma, it inhibited completely the ADP (2 x 10(-5) g/ml)-induced platelet aggregation. It also inhibited the platelet aggregations induced by sodium arachidonate (100 microM), collagen (20 micrograms/ml) and ionophore A-23187 (5 microM)-induced platelet aggregations were not affected significantly by this venom 5'-nucleotidase. In ADP-refractory platelet-rich plasma, the venom 5'-nucleotidase inhibited the platelet aggregations induced by collagen (20 micrograms/ml) or sodium arachidonate (100 microM). The venom 5'-nucleotidase showed a more pronounced inhibitory effect on sodium arachidonate-induced platelet aggregation than creatine phosphate/creatine phosphokinase and apyrase did. No lactate dehydrogenase was released by this venom 5'-nucleotidase, indicating that no platelet lysis occurred. It is concluded that removal of ADP, which is released by these platelet aggregation inducers, and the subsequent accumulation of adenosine are responsible for the inhibitory effect of the venom 5'-nucleotidase on platelet aggregations.
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PMID:Inhibition of platelet aggregation by 5'-nucleotidase purified from Trimeresurus gramineus snake venom. 631 33

Intracellular 5'-nucleotidase involved in membrane circulation in rat hepatocytes is latent, and is protected from inhibition when whole cells are incubated with inhibiting antiserum at 2 degrees C [Stanley, Edwards & Luzio (1980) Biochem. J. 186, 59-69]. These two criteria were used to identify intracellular membrane vesicles containing 5'-nucleotidase on Ficoll density gradients. A sharply defined turbid band containing intracellular 5'-nucleotidase isolated on density gradients was further fractionated by immunoadsorption of plasma-membrane fragments derived from the cell surface of surface-inhibited cells on to an anti-(immunoglobulin G) immunoadsorbent. The resulting non-adsorbed membrane fraction consisted of vesicles of uniform size (approx. 65 nm diam.), but was not identifiable as any known organelle. This fraction could account for approx. 5% of the total cell 5'-nucleotidase activity, and the enzyme activity measured was 55% latent. The fraction had a restricted polypeptide composition but similar phospholipid composition compared with plasma membrane. We suggest that the vesicles observed in this fraction were derived from the endocytic pathway.
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PMID:The isolation of endosome-derived vesicles from rat hepatocytes. 631 38

During growth and maturation of the tapeworm, Hymenolepis diminuta, significant decreases occur in the brush border membrane-bound alkaline phosphatase, phosphodiesterase, 5'-nucleotidase, adenosine triphosphatase and ribonuclease activities. These decreases are accompanied by qualitative and quantitative changes in the polypeptide profiles of the brush border membrane fraction. Gradients of enzymatic activities and polypeptide profiles are also demonstrable when mature tapeworms are cut into pieces and the brush border membrane of each piece analyzed individually. In fully developed tapeworms the enzymatic activities and polypeptide profiles of membrane preparations reflect mainly the contributions of the more mature proglottids; these proglottids constitute most of the tapeworm biomass. The most anterior sections of these fully developed worms are biochemically similar to young, developing worms.
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PMID:Alterations in brush border membrane proteins and membrane-bound enzymes of the tapeworm, Hymenolepis diminuta, during development in the definitive host. 663 65

A membrane fraction denoted N2 upper was isolated from homogenates of rat liver by sucrose gradient centrifugation. This fraction, which was enriched 65-fold over the homogenate in 5'-nucleotidase activity, was used as an immunogen in goats. The antisera obtained contained antibodies to three predominant polypeptides in the N2 upper membrane fraction, as shown by crossed immunoelectrophoresis. These polypeptides had molecular weights of 105,000, 110,000, and 160,000 after recovery from the crossed immunoelectrophoretic gels and are denoted PM105, PM110, and PM160. Each was a distinct polypeptide, as shown by the distinct peptide patterns resulting from limited proteolysis in the presence of detergents. The three polypeptides were synthesized by primary cultures of hepatocytes and were externally oriented at the surface of these cells, as shown by their accessibility in situ to iodination catalyzed by lactoperoxidase. They were not detectable in the serum by crossed immunoelectrophoresis. The three antigens were present at very low (PM110) or nondetectable (PM105, PM160) concentrations in intracellular membrane fractions derived from the Golgi and smooth and rough endoplasmic reticulum of liver. The antigens also were reduced in concentration in a plasma membrane fraction most likely derived from the sinusoidal surface of the hepatocyte. The three membrane antigens bind to concanavalin A; hence, they are probably glycoprotein constituents of a discrete domain of the hepatocyte plasma membrane. Immune complexes were isolated after crossed immunoelectrophoresis and injected into rabbits. Each of the antisera obtained was reactive to one of the membrane polypeptides. Sections of fixed rat livers were reacted with each of the antibodies and then the primary antibody was localized by indirect immunocytochemical methods using horseradish peroxidase or colloidal gold as labels. Each of the three antigens was localized by this method to the bile canalicular domain of the hepatocyte plasma membrane.
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PMID:Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane. 664 79

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5'-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.
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PMID:Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue. 672 59


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