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)

Plasma membranes isolated from Yoshida ascites hepatoma AH-130 by a modification of the method of T.K. Ray (Biochim. Biophys. Acta 196:1, 1970), were subfractionated into three fractions having densities (d) 1.12, 1.14 and 1.16 by discontinuous sucrose density-gradient. Membrane subfractions were characterized by electron-microscopy, by assay of marker enzymes and by lipid composition. All subfractions appeared to be essentially free from whole mitochondria, lysosomes and nuclei. Subfraction d 1.16 had the highest 5'-nucleotidase, Mg++-ATPase and (Na+ +K+)-ATPase activities; cytochrome c oxidase was undetectable in any fraction and glucose-6-phosphatase was measurable only in fraction d 1.14 and 1.16. Cyclic AMP phosphodiesterase was nearly equally distributed in the fractions. Adenylate cyclase, 5'-nucleotidase and Mg++-ATPase activities of tumor membrane were lower with respect to liver plasma membrane, while cyclic AMP phosphodiesterase and (Na" +K+)-ATPase were found to have similar activities in the two membrane preparations. With respect to liver membrane, hepatoma membrane contained a higher amount of glycolipids and a higher amount of phospholipids accounted for mainly by sphingomyelin, phosphatidylserine and phosphatidic acid. The possible significance of the decrease of adenylate activity in the hepatoma membrane is briefly discussed.
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PMID:Isolation and characterization of the plasma membrane from Yoshida hepatoma cells. 16 55

From a homogenate of rabbit colon muscle subcellular fractions were isolated by differential centrifugation. The crude microsomal fraction could be separated into subfractions, a fraction of vesicular microsomes at 35% sucrose, a fraction containing sarcolemma, mitochondrial fragments and microsomal vesicles at 35--45% sucrose and a small protein fraction at 45--55% sucrose. Their biochemical properties and their morphological characterization were investigated. The cholesterol and the phospholipid content was equally distributed between the microsomal fractions 35% and 35--45% while the RNA was localized to the mitochondria and the microsomal fraction 35%. The enzyme cytochrome c oxidase was found to be concentrated in the mitochondria while a high contamination was found in the microsomal fractions 35--45%. The NADH-oxidase activity was highest in the 35% fraction and the 5'-nucleotidase activity in the 40,000 X g supernatant. The microsomal subfractions contained the enzymes ATPase, adenylate cyclase and phosphodiesterase. In the 35% fraction Ca stimulated the hydrolysis of ATP. The binding of [3H]-ouabain and the incorporation of [3H]-leucine was most pronounced in the 35% fraction. In a K+-free Krebs Ringer medium the binding of the glucoside was stimulated in all the fractions. From these results we concluded that the fraction 35% sucrose may be mainly derived from the endoplasmic reticulum and the plasma membrane while the 35--45% originates from the plasma membrane, mitochondria and to a lesser extent the endoplasmic reticulum.
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PMID:Biochemical and morphological characterization of subcellular fractions isolated from rabbit colon muscle. 20 90

Mitochondrial and microsomal fractions were isolated from guinea pig myocardium by differential pelleting. The mitochondrial fraction was subjected to analytical subfractionation by sucrose density gradient centrifugation and the gradient fractions assayed for marker enzymes for the various mitochondrial compartments, viz outer membrane (monoamine oxidase), intermembranous space (adenylate kinase), inner membrane (Mg2+-dependent ATPase and cytochrome c oxidase) and mitochondrial matrix (malate dehydrogenase), and for creatine kinase. Both creatine kinase and adenylate kinase were released by suspending the mitochondria in 50 mmol . litre-1 sodium phosphate buffer. Sonication or disruption with the detergent, digitonin released the adenylate kinase but the creatine kinase remained associated with the inner membranes. Subsequent salt treatment desorbed the creatine kinase from these membranes. It is concluded that creatine kinase is located to the outer aspect of the inner mitochondrial membrane. Analytical subfractionation of the microsomal fraction clearly resolved markers for the sarcolemma (5'-nucleotidase), outer mitochondrial membrane (monoamine oxidase) and endoplasmic reticulum (neutral alpha-glucosidase and RNA). Creatine kinase was localised in the endoplasmic reticulum particularly the smooth membranes.
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PMID:Sub-mitochondrial and sub-microsomal distribution of creatine kinase in guinea pig myocardium. 51 58

Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5'-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium-dependent ATPase and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific acivity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.
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PMID:Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets. 79 56

The controversial subject of the subcellular location of myocardial adenosine production was studied employing density gradient fractionation of heart muscle combined with a novel method for analyzing distribution profiles based on multiple regression (correlation) analysis. Bungarotoxin binding, N-acetyl-beta-D-glucosaminidase, cytochrome c oxidase, NADPH-dependent cytochrome c reductase and lactate dehydrogenase were used as markers for the plasma membrane, lysosomes, mitochondria, sarcoplasmic reticulum and cytosol, respectively. The normalized distribution frequencies (fraction of total) of 5'-nucleotidase in mitochondria, lysosomes, plasma membranes, sarcoplasmic reticulum and cytosol in the 50 x g supernatant of total homogenate of heart muscle were found to be 0, 0.25, 0.44, 0.08 and 0.23, respectively. To increase the resolution power of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied, in which the normalized distribution of 5'-nucleotidase in the homogenate was 0, 0.16 and 0.84 in mitochondria, plasma membranes and lysosomes, respectively. This mainly lysosomal 5'-nucleotidase activity was 61% inhibited by the alpha,beta-methylene analog of ADP, indicating that although the latter has been considered specific to the plasma membrane enzyme, it also inhibits the lysosomal enzyme. The intercellular distribution of 5'-nucleotidase was not studied, but the lack of this enzyme in the mitochondria indicate that the adenosine production observed during mitochondrial AMP production, e.g. during acetate oxidation in intact heart muscle, must involve AMP transport out from the mitochondria.
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PMID:Subcellular distribution of myocardial 5'-nucleotidase. 223 47

We have undertaken the analytical fractionation of epithelial cells from toad urinary bladder, a tissue extensively used to study epithelial transport of ions and water. In an attempt to establish markers for the main subcellular organelles, a number of enzymes were assayed in cell homogenates. The nearly ubiquitous plasma membrane marker 5'-nucleotidase, and the transferases that donate N-acetylglucosaminyl, galactosyl, and sialyl residues to glycoproteins and glycolipids in the Golgi complex were not detectable. Glucose-6-phosphatase activity was low in relation to that of nonspecific phosphatases and, therefore, not suitable for identifying the endoplasmic reticulum. Like the cytosolic enzyme lactate, dehydrogenase, catalase was essentially found in the high-speed supernatant, with a noteworthy part of aminopeptidase (substrate, leucyl-beta-naphthylamide) and NAD glycohydrolase. Other enzymes, including cytochrome c oxidase, acid phosphatase, acid N-acetyl-beta-glucosaminidase, alkaline phosphatase, alkaline phosphodiesterase I, nucleoside diphosphatase (substrate ADP), oligomycin-resistant Mg++-ATPase, and mannosyltransferase (acceptor, dolichylphosphate) were fairly active and largely sedimentable. After differential centrifugation, cytochrome oxidase, acid phosphatase, and acid N-acetyl-beta-glucosaminidase were typically associated with the large granule fraction, whereas the other sedimentable enzymes exhibited a broad distribution profile overlapping the nuclear, large granule, and microsome fractions. Their behavior in density equilibrium centrifugation is examined in a companion paper.
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PMID:Subcellular fractionation of epithelial cells from toad urinary bladder. 1. Assay of marker enzymes and differential centrifugation. 250 71

The distribution of [3H]leukotriene D4 [( 3H]LTD4) receptors in subcellular membrane fractions obtained from sheep tracheal smooth muscle was studied. Using differential centrifugation and discontinuous sucrose density gradient centrifugation, the subcellular membranes were separated into six fractions. The [3H]LTD4 receptor distribution profile in these fractions correlated with markers for the plasma membrane (5'-nucleotidase and alkaline phosphodiesterase) and did not correlate with markers for the mitochondria (cytochrome c oxidase and succinate-dependent cytochrome c reductase). The dissociation constant (Kd) and maximum number of binding sites (Bmax) for [3H]LTD4 binding to the receptors in the crude mixture of membranes (PII) were 0.38 +/- 0.2 nM and 77 +/- 14 fmol/mg of protein, respectively. The Kd and Bmax of [3H]LTD4 binding to the receptors in the plasma membrane-enriched fraction (FII) were 0.40 +/- 0.2 nM and 268 +/- 46 fmol/mg of protein, respectively. The specificity profile of the [3H]LTD4 receptors in the plasma membrane-enriched fraction was equivalent to that observed in the crude membrane and correlated with the agonist myotonic activities in the smooth muscle contraction assay system. Furthermore, the binding of [3H]LTD4 to the plasma membrane receptors was modulated by guanine nucleotides in a manner analogous to that observed in crude membranes, suggesting that agonist interaction with the receptors was regulated by guanine nucleotide binding protein. These results suggest that, in sheep tracheal smooth muscle, the plasma membrane is the primary location of specific LTD4 receptors.
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PMID:Subcellular localization of leukotriene D4 receptors in sheep tracheal smooth muscle. 284 53

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.
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PMID:Subcellular fractionation of pig coronary artery smooth muscle. 299 88

To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.
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PMID:Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3-125I)-labelled neurotensin binding. 302 74

This communication presents the results obtained in tubular aggregates of 24 enzyme histochemical techniques for demonstrating activity of oxidoreductases, transferases, hydrolases and isomerases. The activity characteristics of the tubular aggregates in m. gluteus medius of 18 patients with diseases of the neuromuscular system were almost identical. A high activity of the mitochondrial enzymes, NADPH: tetrazolium oxidoreductase, NADH:tetrazolium oxidoreductase and cytochrome c oxidase, could be shown in the pathological structures, whereas the activity of the mitochondrial enzymes, glycerol-3-phosphate:menadione oxidoreductase, succinate:PMS oxidoreductase, malate:NAD+ oxidoreductase and isocitrate:NAD+ oxidoreductase, and the partial mitochondrial enzymes, malate:NADP+ oxidoreductase and isocitrate:NADP+ oxidoreductase, was very slight or even absent. There was a moderate to strong activity of the glycolytic enzymes lactate:NAD+ oxidoreductase, glyceraldehyde-3-phosphate:NAD+ oxidoreductase, phosphofructokinase, phosphoglucomutase and glucose phosphate isomerase. In contrast, the activity of alpha-glucan phosphorylase was slight. The activity of phosphogluconate:NADP+ oxidoreductase, glucose-6-phosphate:NADP+ oxidoreductase and 5'-nucleotidase was slight, whereas there was no activity of myosin ATPase and mitochondrial ATPase, acid phosphatase or alkaline phosphatase. The high activity of AMP-deaminase was very striking. The activity of peroxidase was moderate. Results obtained with adsorption studies point to adsorption of some of the enzymes studied to the tubular aggregates in vivo and this phenomenon very probably determined the histochemical characteristics of these structures.
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PMID:Histochemical features of tubular aggregates in diseased human skeletal muscle fibres. 317 98


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