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 membrane vesicles were isolated from a subline of L929 mouse fibroblasts grown on defined medium in the absence of serum. These vesicles were not significantly contaminated by mitochondria or endoplasmic reticulum. The isolation procedure, a modification of that originally developed by McKeel and Jarett (McKeel, D.W., and Jarett, L. (1970) J. Cell Biol. 44, 417-432) employs mechanical homogenization in isotonic medium followed by differential centrifugation. The resultant plasma membrane vesicles take up radioactivity when exposed to uniformly labeled nucleosides. Two subfractions of the plasma membrane were isolated, distinguished by their differing activity of 5'-nucleotidase and (Na+,K+)-stimulated ATPase, two well known plasma membrane enzyme markers. Uptake of nucleoside radioactivity was extensively studied in one subfraction; it was linear with time and membrane concentration over ranges used for the studies. Apparent Km values for uptake of radioactivity from adenosine, inosine, and uridine were 7.1 +/- 26 muM, respectively. Uptake of radioactivity from all three nucleosides exhibits a broad pH optimum from pH 7 to pH 9, but falls off rapidly at lower pH. N-Ethylmaleimide was an effective inhibitor of uptake of radioactivity from all three nucleosides; uptake of radioactivity from uridine is more sensitive than uptake of radioactivity from the purine nucleosides. Adenosine inhibited uptake of radioactivity from inosine more than from uridine. Inosine inhibited the uptake of radioactivity from adenosine, but uridine did not. Caffeine and 6-methylaminopurine riboside (6-N-methyladenosine differentially inhibit uptake of radioactivity from adenosine and inosine, and thus the vesicles apparently possess seperate transport systems for uptake of radioactivity from purine nucleosides and from uridine.
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PMID:Transport mechanisms in isolated plasma membranes. Nucleoside processing by membrane vesicles from mouse fibroblast cells grown in defined medium. 0 4

Membrane vesicles can be prepared from murine lymphoid cells by nitrogen cavitation and fractionated by sedimentation through nonlinear sucrose density gradients. Two subpopulations of membrane vesicles, PMI and PMII, can be distinguished on the basis of sedimentation rate. The subcellular distribution of adenylate and guanylate cyclases in these membrane subpopulations have been compared with the distribution of a number of marker enzymes. Approximately 20-30% of the total adenylate and guanylate cyclase activity is located at the top of the sucrose gradient (soluble enzyme), the remainder of the activity being distributed in the PMI and PMII fractions (membrane-bound enzyme). More than 90% of the 5'-nucleotidase and NADH oxidase activities detected in lymphoid cell homogenates are located in PMI and PMII fractions, whereas succinate cytochrome c reductase activity is detected only in the PMII fractions. In addition, beta-galactosidase activity is distributed in the soluble and PMII fractions of the sucrose density gradients. On the basis of the fractionation patterns of these various enzyme activities, it appears that PMI fractions contain vesicles of plasma membrane and endoplasmic reticulum, whereas PMII fractions contain mitochondria, lysomes, and plasma membrane vesicles. Approximately 30-40% of the adenylate and guanylate cyclase activities in PMII can be converted to a PMI-like form following dialysis and resedimentation through a second nonlinear sucrose gradient. Adenylate and guanulate cyclases can be distinguished on the basis of sensitivity to nonionic detergents.
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PMID:The subcellular distribution of adenylate and guanylate cyclases in murine lymphoid cells. 0 90

Centrifugation of homogenates of bovine retinas to isopycnic equilibrium in sucrose density gradients yielded three partially overlapping bands of particles which were, in the order of increasing density: (a) photoreceptor cell (rod) outer segments; (b) plasma membranes, lysosomes, and large fragments of endoplasmic reticulum; and (c) mitochondria. The only enzyme activity investigated which had a peak coinciding only with outer segment fractions was guanylate cyclase. Enzyme activities with peaks in both the outer segment and denser fractions included 5'-nucleotidase and cyclic GMP phosphodiesterase. Enzyme activities with peaks only in the denser fractions included sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase), adenylate cyclase, cyclic AMP phosphodiesterase, beta-glucosidase, beta-galactosidase, and succinate-dependent cytochrome c reductase. These results suggest that some of the activities once thought to be present in rod outer segments are actually present in particles from elsewhere in the retina which contaminate rod outer segment preparations.
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PMID:Distribution of enzyme activities in subcellular fractions of bovine retina. 0 65

A method is described for the localization and characterization of phospholipases A1 and A2 (EC3.1.1.4) in Krebs II ascites cells, particularly in the plasma membranes. Cells were lysed with a Dounce homogenizer in an isotonic sucrose medium. Plasma membranes sediment with mitochondria and lysosomes during subcellular fractionation and are finally isolated on a continuous sucrose gradient. The membranes are localized at two levels in the gradient, at densities of 1.06 and 1.15, in which 5'-nucleotidase (EC 3.1.3.5) activity exhibits a 9- and 21-fold purification, respectively. Total contamination by endoplasmic reticulum, lysosomes, and mitochondria is 17 percent for the low-density membrane fraction and 25 percent for the high-density fraction. The phospholipases A present in Krebs II cells are active at pH 4.0 and pH 7.5. At the 2 pH values, they have A1 and A2 specificities. The intracellular distribution of acidic forms is comparable to that of acid phosphatase (EC 3.1.3.1), while neutral forms are localized like lactate dehydrogenase (EC 1.1.1.27). A small proportion of neutral phospholipase A2 has the same repartition on the sucrose gradient as nicotinamide adenine dinucleotide diaphorase (EF 1.6.4.3), an endoplasmic reticulum marker, and as 5'-nucleotidase, a plasma membrane marker.
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PMID:Phospholipases A1 and A2 in subcellular fractions and plasma membranes of Krebs II ascites cells. 2 44

Portions of closed jejunal biopsies from the dog were homogenised and their organelles separated by isopycnic centrifugation on continuous sucrose density gradients. The distributions of marker enzymes for the principal organelles were determined using highly sensitive assay procedures. The following organelles, with assayed marker enzymes and modal densities between brackets were characterised: peroxisomes (catalase, 1.21); brush borders (zinc-resistant alpha-glucosidase, leucyl-beta-naphthyl-amidase, gamma-glutamyl transferase, alkaline phosphatase, 1.20); lysosomes (N-acetyl-beta-glucosaminidase, alpha-mannosidase, 1.19); mitochondria (malate dehydrogenase, 1.18); endoplasmic reticulum (Tris-resistant alpha-glucosidase, 1.16); basal-lateral membranes (5'-nucleotidase, 1.11) and cytosol (lactate dehydrogenase). Homogenisation in isotonic sucrose containing digitonin (0.12 mmol/litre) selectively disrupted lysosomes and increased the equilibrium density of brush border and basal-lateral membranes. This procedure will be used to study the subcellular pathology of naturally occurring intestinal disease in the dog.
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PMID:Subcellular fractionation studies on peroral jejunal biopsies from the dog. 3 Jan 25

The localization of gamma-glutamyltransferase activity in guinea pig liver was studied after subcellular fractionation. The enzyme activity was essentially connected with plasma membranes whereas only low activity was found in the endoplasmic reticulum. A similar activity distribution was demonstrated for 5'-nucleotidase. Highest specific activity of gamma-glutamyltransferase was found in plasma membranes enriched in bile canaliculi. In this fraction the specific activity was 35 times greater than the specific activity of the total homogenate, a value similar to the relative specific activity of (Na+,K+)-ATPase. More than 90% of the total gamma-glutamyltransferase activity in guinea pig liver was connected with parenchymal cells and the enzyme seemed to have an outside orientation. Animals treated with phenobarbital showed moderate increased in gamma-glutamyltransferase activity in serum and liver, whereas high activities were found in most bile samples. No particular liver subfraction showed substantial accumulation of gamma-glutamyltransferase activity. The present findings do not support the suggested use of serum gamma-glutamyltransferase measurements as a direct index of "microsomal enzyme induction".
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PMID:Subcellular localization of gamma-glutamyltransferase activity in guinea pig liver. Effect of phenobarbital on the enzyme activity levels. 3 6

Large amounts of injected radiolabeled low density lipoproteins have been found by others to accumulate primarily in the liver and studies in various types of isolated cells, including hepatocytes, have indicated the presence of specific cell membrane recognition sites for lipoproteins. In the present studies, the high affinity binding of radiolabeled low density lipoproteins ([125I]LDL, d 1.020--1.063 g/mL) was measured in the major subcellular fractions of porcine liver homogenates. The nuclear and mitochondrial fractions were 1.9- and 1.4-fold enriched in binding activity with respect to unfractionated homogenates and contained 15% and 12% of the total binding activity, respectively. The microsomes, which contained most of the plasma membranes and endoplasmic reticulum, were approximately 4-fold enriched in binding and contained 73% of the binding activity. Microsomal subfractions obtained by differential homogenization and centrifugation procedures were 5.6--7.0-fold enriched in LDL binding and contained 54--58% of the homogenate binding activity. They were separated by discontinuous sucrose density gradient centrifugation into fractions which contained "light" and "heavy" plasma membranes and endoplasmic reticulum. The heavy membrane fraction was 2--4 fold in binding with respect to the parent microsomes (16--22 fold with respect to the homogenate). There was no enrichment of binding activity in the other two fractions. Two plasma membrane "marker" enzymes, nucleotide pyrophosphatase and 5'-nucleotidase, were also followed. Of the two, binding in the sucrose density gradient subfractions most closely followed nucleotide pyrophosphatase, which was also most highly enriched (3.2--3.3-fold) in the heavy membrane fraction, but did not follow it exactly. The enzyme was 2-fold richer in the light membranes than in the parent microsomes, though the light membrane binding activity was only 0.4--1.4 times that of the parent microsomes. High affinity binding was time and temperature dependent, saturable, and inhibited by unlabeled low density lipoproteins but not by unrelated proteins. Binding was stimulated 2--3 fold Ca2+, was not affected by treatment with Pronase or trypsin and was inhibited by low concentrations of phospholipids and high density lipoproteins (HDL). Heparin-Mn2+ treatment of HDL did not affect its ability to inhibit [125I] LDL binding. The LDL recognition site was distinct from the liver membrane asialoglycoprotein receptor; LDL binding was not inhibited by desialidated fetuin. We conclude that porcine liver contains a high affinity binding site that recognizes features common to both pig low density and high density lipoproteins. Further studies may elucidate the significance of this binding site in lipoprotein metabolism.
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PMID:Isolation of a porcine liver plasma membrane fraction that binds low density lipoproteins. 8 56

Plasma membranes from normal, full-term human placental trophoblast have been isolated by a new procedure. The method depends upon isopycnic zonal centrifugation using linear sucrose/Ficoll density gradients. Enrichment of plasma membrane marker enzymes with respect to trophoblast homogenate is found in two distinct peaks (designated B and D) of the fractionated effluent recovered from the rotor. Fraction B is enriched with membrane-bound alkaline phosphatase and 5'-nucleotidase, but not with (Na+, K+)-ATPase of F(-)-stimulated adenylate cyclase. It is suggested that this material is derived from the maternal-facing microvillous plasma membrane. Fraction D, enriched with (Na+, K+)-ATPase, F(-)-stimulated adenylate cyclase and, to a smaller extent, with 5'-nucleotidase and alkaline phosphatase is, by exclusion, proposed to be derived from the fetal-facing basal plasma membrane. Both plasma membrane fractions are shown to be free of appreciable contamination, using specific markers for endoplasmic reticulum, mitochondria, nuclei and lysosomes. The separation of the two membrane fractions is shown to depend both upon these membranes forming closed vesicles during homogenization and upon the buoyant densities of such vesicles differing in such a way that microvillous plasma membranes band at a lower density than basal plasma membranes. No separation of the membranes is achieved in gradients in which the vesicles are collapsed.
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PMID:Separation of the microvillous (maternal) from the basal (fetal) plasma membrane of human term placenta: methods and physiological significance of marker enzyme distribution. 9 48

Three lysosomal glycosidases, beta-glucuronidase (EC 3.2.1.31), beta-galactosidase (EC 3.2.1.23), and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) have been investigated in bile that was freshly collected from rats through a complete bile fistula. Assay conditions have been established on the basis of appropriate kinetic studies. The biliary excretion patterns for these enzymes were found to vary considerably from rat to rat during the 24-h collection period. In a given animal, however, the three hydrolases were excreted in parallel and showed a gradual increase in activity with time, most marked after 10- 12 h of collection. 24-h biliary outputs of the three hydrolases averaged congruent with3% of their respective contents in total liver, and bile diversion had no effect on hepatic glycosidase activity or total protein content. Other enzymes known to be associated primarily with mitochondria, endoplasmic reticulum, and cell sap were also detected in bile, generally in smaller amounts. The biliary excretion of the plasma membrane markers, alkaline phosphodiesterase I and 5'-nucleotidase, however, was comparable to that of the lysosomal hydrolases. Biliary excretion of total protein was relatively constant and corresponded to 3.0% of the total hepatic protein content per day, whereas biliary bile acid secretion decreased during the first 12 h and then remained constant. Exocytic bulk discharge of hepatocyte lysosomes is proposed as the most likely mechanism for the biliary excretion of lysosomal enzymes. These results call attention to the possible pathophysiologic significance of biliary excretion of hepatic lysosomal contents as a means of residue disposal.
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PMID:Coordinate secretion of acid hydrolases in rat bile. 11 27

Differential centrifugation was applied to adult and foetal liver of monkey. Obtained fractions were: F1 (800 X g); F2 (12 500 X g); F3 (200 000 X g); and cell sap. Analysis of chemical compounds of these fractions shows that: (1) adult and foetal nucleic acids levels are similar; (2) there are more proteins in adult than in foetal hepatocytes; (3) most of the glycogen is located in F3; the foetal level is twenty times higher than the adult level. Plasma membrane enzymes (5'-nucleotidase, adenylate cyclase) show a nucleomicrosomic distribution. The distribution of alkaline phosphatase is not significant. Mitochondrial enzymes (monoamine oxydase, succinate cytochrome c reductase, cytochrome oxydase) are enriched in F2 without any sedimentation in F3. There is more malate dehydrogenase liberated in cell sap during foetal liver fractionation. This indicates the foetal mitochondria are more sensitive to the homogenisation method. Lysosomal enzymes (acid phosphatase, N-acetylglucosaminidase) are enriched in F2. The same observation for N-acetylglucosaminidase as for malate dehydrogenase leads to the same conclusion for foetal lysosomes. Endoplasmic reticulum and Golgi enzymes (glucose-6-phosphatase and related phosphotransferase activity, NADPH-cytochrome c reductase and sialytransferase) are much enriched in F3. Thus this fraction F3 is pure enough to allow the observation of the modification produced on endoplasmic reticulum and Golgi apparatus during foetal and neonatal development.
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PMID:[Comparative study of microsomal enzymic activities in adult and foetal monkey hepatocytes (author's transl)]. 11 30


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