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

Heterosis (hybrid vigor) for brain myelin content has been examined in detail in (C57BL/6J x DBA/2J)F1 hybrid mice at 17 days of age. The amount of myelin isolated from the F1 hybrid brain is greater than that isolated from either parental strain. In addition, the total protein content in the myelin of the three genotypes showed the following trend: F1 greater than DBA greater than C57. However, no discernible differences in myelin protein compositions could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the whole brain for several myelin-associated constituents such as GM1 ganglioside, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), 5'-nucleotidase, and carbonic anhydrase indicated that heterosis exists for these components. No heterosis was found for such nonmyelin constituents as gangliosides GD1a, GT, GQ, RNA, DNA, and choline acetyltransferase. A developmental study of the whole brain CNPase indicated that the heterotic effect was greatest during the most active period of myelination (17-30 days). We conclude that the heterotic effect is specific for myelin content and is probably the result of an accelerated myelin synthesis. The heterotic effect should have great potential as a new model for studying aspects of myelinogenesis.
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PMID:Biochemical study of heterosis for brain myelin content in mice. 618 36

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

A procedure was developed to purify plasma membranes from cardiac tissue. Canine ventricular tissue was homogenized relatively gently in hypotonic buffer, extracted briefly with 0.15 M sodium perchlorate at 4 degrees C, and membranes were collected in an intermediate pellet by centrifugation at 27,500 X gmax. At this stage, most contractile proteins were removed (although Z-band structures were apparent), and the recoveries of Na+, K+-ATPase, beta-adrenoreceptors and 5'-nucleotidase were 40--50%. Treatment of these particles with 0.5% dextran sulfate and continuous density gradient centrifugation resulted in the separation of membranes having 111 and 3.6 pmol/mg protein specific binding sites for [3H] ouabain and [3H] dihydroalprenolol, respectively. Electron micrographs showed medium-sized vesicles. 5'-Nucleotidase was also markedly purified but could be partially separated from the other plasma membrane markers in continuous sucrose gradients. Enzymes associated with mitochondria, lysosomes, and endoplasmic reticulum were efficiently removed. SDS-polyacrylamide gels showed six major and many minor bands. Adenylate cyclase remained responsive to hormones and Gpp(NH)p, but little purification was apparent. The membrane preparation has proved useful for studies of a nucleotide-binding protein associated with cyclase activation.
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PMID:Purification and partial characterization of cardiac plasma membranes rich in beta-adrenoreceptors. 624 6

The growth of mouse mastocytoma P-815 cells in culture (37 degrees, 42 hr) was inhibited by exogenous adenosine (0.2 to 1.0 mM) and more effectively by AMP (0.01 to 0.1 mM), but not by adenine. The inhibited growth (a 25% inhibition by 0.5 mM adenosine and a 80% inhibition by 0.25 mM AMP) was restored to a near control level by the addition of uridine (0.5 mM) to the medium. The pretreatment (37 degrees, 3 hr) of the cells with adenosine or AMP caused a 60% inhibition of incorporation (37 degrees, 2 hr) of [U-14C]aspartate into uracil nucleotides, accumulating 14C-orotate and orotidine. Both dipyridamole, an inhibitor of adenosine uptake, and exogenous adenosine deaminase suppressed the growth inhibition induced by not only adenosine but also AMP. 2-Chloroadenosine, which is resistant to the action of adenosine deaminase, was a more potent growth inhibitor, while 3'AMP and 2'-AMP, which are not hydrolyzed to adenosine by membrane 5'-nucleotidase, were ineffective. Adenosine 5'-sulfate and other 5'-substituted adenosines were also ineffective. These observations indicate that AMP inhibits the growth of mastocytoma P-815 cells as a result of its continuous conversion to adenosine and a constant exposure of the cells to a low concentration of adenosine which readily permeates the cell membrane. In addition, adenosine, AMP and their agarose-linked forms rapidly (37 degrees, 20 min) elevated cellular levels of cAMP. This effect was not suppressed by dipyridamole. Apparently adenosine and AMP also act extracellularly for growth inhibition by regulating cAMP levels.
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PMID:Effect of adenosine and adenosine 5'-monophosphate on cell division of cultured mastocytoma P-815 cells. 625 14

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

The ability of nonionic detergents to solubilize the membrane-bound enzymes of the brush-border plasma membrane of Hymenolepis diminuta was investigated. Of the detergents tested (Triton X-100, Tween 80, Brij 35, Lubrol PX and WX, W-1, and beta-octyl-D-glucoside), only Triton was an effective solubilizing agent. Optimal solubilization was achieved by incubating an isolated fraction of the brush-border membrane in the presence of 1% Triton X-100 for 60 min at 37 C, followed by centrifugation at 100,000 g for 60 min at 25 C. This treatment resulted in solubilization of 94% of the alkaline phosphohydrolase, 91% of the phosphodiesterase and ribonuclease, and 88% of the 5'-nucleotidase activities. The pH optima for enzymes solubilized in nonionic and ionic detergents (Triton and sodium dodecyl sulfate, respectively) did not differ. Isoelectric focusing of the Triton-solubilized material demonstrated the presence of at least 14 polypeptides, a majority of which had isoelectric points below pH 7.
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PMID:Solubilization of the membrane-bound enzymes of the brush-border plasma membrane of Hymenolepis diminuta (Cestoda) using nonionic detergents. 628 6

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.
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PMID:Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins. 629 Feb 20

Two substrate proteins for cAMP-dependent protein kinase detected in a rat heart sarcolemma preparation displayed molecular weights of 24,000 and 9000 in sodium dodecyl sulfate gels and were shown to be interconvertible. The 9000-dalton protein could readily be separated from other low molecular weight phosphoproteins (mol. wt. 14,000 and 7000) by the use of 15% polyacrylamide gels. In addition to an endogenous cAMP-dependent protein kinase the membrane preparation also contained a protein-phosphorylation system that required Ca2+ and calmodulin. It appeared that both 24,000- and 55,000-dalton proteins were substrates for the endogenous Ca2+- and calmodulin-dependent protein kinase. Contaminating sarcoplasmic reticulum vesicles, first loaded with calcium oxalate, could be separated from the enriched sarcolemma preparation by sucrose gradient centrifugation. The separation was confirmed by comparative analysis of 5'-nucleotidase, Na+ -Ca2+ antiporter, and (Ca2+ + Mg2+)-dependent ATPase activities and by determination of gel electrophoretic (phospho)protein composition, sialic acid, cholesterol, and phospholipid contents. The 24,000-dalton phosphoprotein complex was equally distributed between sarcolemmal and sarcoplasmic reticulum fractions, whereas the 55,000- and 7000-dalton proteins were predominantly found in the sarcolemmal fraction. The 24,000-dalton protein was most likely phospholamban, because no other phosphoprotein was found in the 20,000 molecular weight range.
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PMID:Phosphorylation of low-molecular-weight proteins in preparations of rat heart sarcolemma and sarcoplasmic reticulum. 630 73

Sarcolemmal vesicles isolated from relaxed rat cardiac ventricles were 120-fold enriched in (Na+ + K+)-ATPase and 5'-nucleotidase activities (final recoveries, 50%). The alpha and beta chains of the former enzyme were visualized by the immunological approach. Inside-out sarcolemmal vesicles were isolated by affinity chromatography on immobilized concanavalin A. The yield of membranes was 0.45 mg of protein/g of muscle. The orientation of the unbound vesicles was studied by the increased accessibility of sarcolemma outer face markers (ouabain- and K+-binding sites, 5'-nucleotidase, and sialic acids) with permeability-increasing treatments: freeze-thaw cycles, sodium dodecyl sulfate, methanol, and valinomycin. The total ATP hydrolysis remained constant with a conversion of ouabain-insensitive activity into an ouabain-sensitive one. These agents caused a parallel increase in the ouabain sensitivity, the number of [3H]ouabain-binding sites, the monovalent cation stimulation of ATPase, and the 5'-nucleotidase activity. Valinomycin revealed that most vesicles were sealed to sequestered and exogenous K+. Inside-out vesicles were 80% pure in sidedness and sealing. The affinity chromatography did not affect the (Na+ + K+)-ATPase activity (200 mumol of product/mg of protein/h). This model of sarcolemma vesicles offers a new tool for ion transport studies.
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PMID:Isolation of impermeable inside-out vesicles from an enriched sarcolemma fraction of rat heart. 630 49


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