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)

Homogenates of control and diet-induced atherosclerotic aortas of rabbit were prepared and the levels of DNA, protein, free and esterified cholesterol, and six enzymes known to be associated with various subcellular organelles [N-acetyl-beta-glucosaminidase, beta-galactosidase (lysosomes); cytochrome oxidase (mitochondria); neutral alpha-glucosidase (endoplasmic reticulum); 5'-nucleotidase (plasma membrane); catalase (peroxisomes)] were compared between control and atherosclerotic preparations. The levels of prostaglandins I2, E2, and F2 alpha, based on DNA, also were measured by radioimmunoassay. Atherosclerotic aortas were significantly enriched in catalase activity (440%) and in each of the acid hydrolases (395 and 630%), based on DNA, as well as in free (630%) and esterified cholesterol (930%), based on tissue wet weight, compared to control aortas. The control level of prostaglandin I2 was 10-fold higher than that of prostaglandin E2, which was 3-fold higher than that of prostaglandin F 2 alpha. Prostaglandin I2 doubled in amount with advanced atherosclerosis, while prostaglandin E2 increased over 10-fold, resulting in twice the amount of prostaglandin I2 than E2 in advanced atherosclerosis; the level of prostaglandin F2 alpha did not appear to change significantly with atherosclerosis. Increased levels of prostaglandins I2 and E2 were correlated significantly with increased aortic total cholesterol content (based on DNA) but not increased serum cholesterol levels. N-Acetyl-beta-glucosaminidase activity also was correlated significantly to aortic total cholesterol content and beta-galactosidase activity, as well as to the level of prostaglandin I2; in contrast, N-acetyl-beta-glucosaminidase was not significantly correlated to prostaglandin E2. The association of prostaglandins I2 and E2 with aortic total cholesterol suggests the participation of prostaglandins in the response of arterial cells to lipid accumulation in atherosclerosis. The specific association of aortic prostaglandin I2 level and N-acetyl-beta-glucosaminidase activity further suggests a possible role for this prostaglandin during arterial intralysosomal cholesterol accumulation.
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PMID:Arterial prostaglandins and lysosomal function during atherogenesis. I. Homogenates of diet-induced atherosclerotic aortas of rabbit. 389 3

Rat mesenteric artery microsomes were previously reported to degrade 125I-angiotensin II (AII). It is now shown here that washing the membranes with EDTA and including EGTA in the assay media reduces the 125I-AII degradation to very low levels without reducing the specific binding of 125I-AII. Using EDTA wash and including 5 mM MgCl2 and 0.2 mM EGTA the following characteristics of the binding were observed: microscopic association rate constant (k1) = 1.3 to 2.2 X 10(5) M-1 s-1, microscopic dissociation rate constant (k-1) = 3.8 to 6.4 X 10(-4) s-1, equilibrium dissociation constant (Kd) = 1.8 nM and number of binding sites (Bmax) = 193 fmol/mg protein. The subcellular distribution of the specific binding of 125I-AII at 0.16 nM and 1.63 nM was studied along with the distribution of the marker enzymes. The specific binding paralleled the plasma membrane marker (5'-nucleotidase), but not the putative endoplasmic reticulum marker (NADPH-cyt. c-reductase) or the inner mitochondrial marker (cyt. c-oxidase). Thus the binding to the plasma membrane-enriched fraction F2 occurred with a similar affinity (Kd = 2.2 nM) but with higher number of binding sites (420 fmol/mg protein). This study establishes the 125I-AII binding method suitable for determining the changes in the angiotensin receptor characteristics in the pathophysiology of the vascular smooth muscle.
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PMID:Binding and degradation of angiotensin II by mesenteric artery subcellular membranes. 392 92

The isolation of plasma membrane from human peripheral blood monocytes is described. Monocytes were isolated by centrifugal elutriation, to eliminate an adherence step, thus minimizing functional and surface antigenic alterations to the cells. Monocytes were surface-labelled with a radiolabelled monoclonal antibody, 125I-WVH-1, and then disrupted by nitrogen cavitation. Membranes were separated according to equilibrium buoyant density by isopycnic centrifugation on a sucrose gradient. The subcellular membranes were localized using marker enzymes for the plasma membrane, 5'-nucleotidase and leucine 2-naphthylamidase (leucine aminopeptidase), and for intracellular membranes: galactosyltransferase (Golgi), arylsulfatase C (endoplasmic reticulum), monoamine oxidase (mitochondria), catalase (peroxisomes), beta-hexosaminidase and beta-glucuronidase (lysosomal vesicles) and lactate dehydrogenase (cytosol). The monoclonal antibody 125I-WVH-1 was shown to label the plasma membrane, as judged by known markers, and represents a highly specific trace label, applicable to the use of plasma membrane as an immunogen for monoclonal antibody production. The NAD-splitting enzyme, NAD+ nucleosidase, was detected and its presence on the plasma membrane was demonstrated. The subcellular localization of non-specific esterase in human mononuclear phagocytes is controversial. No evidence was found for alpha-naphthyl acetate esterase activity on the plasma membrane or in lysosomal vesicles. However, a membrane-bound esterase in fractions with properties similar to the smooth endoplasmic reticulum was detected.
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PMID:Isolation of plasma membrane from human blood monocytes. Subcellular fractionation and marker distribution. 397 89

Bovine anterior pituitary glands were fractionated by differential centrifugation. T4 5'-monodeiodination to T3 was found predominantly in microsomal fractions (M2; 105,000 . g pellet) enriched in glucose-6-phosphatase and 5'-nucleotidase activities. T4 5'-deiodinase activity in M2 fraction was 85.2 fmol T3/min X mg protein and represented an 8.5-fold enrichment over homogenate specific activity (10.6 fmol T3/min . mg protein). Further subcellular localization of the T4 5'-deiodinase was effected by discontinuous sucrose density gradient centrifugation. Maximum T4 5'-deiodinase activity was found in fraction P5 at the interface of densities 1.18/1.20 (200 fmol T3/min . mg protein) and correlated with the profile of glucose-6-phosphatase and not with that of 5'-nucleotidase, the maximum activity of which was recovered in fraction P1 at the interface of densities 1.03/1.12. Electron microscopic examination of the fractions confirmed that P5 contained in excess of 90% rough membranes in contrast to 10% or less in P1. Characterization of T4 5'-deiodinase activity was carried out in M2 preparations. The reaction was thiol dependent, requiring the presence of 50 mM dithiothreitol or more (Km, 38 mM), with a maximum velocity of 55-150 fmol T3/min . mg protein (n = 8). Enzyme activity was substrate dependent, with a Km for T4 between 35-70 nM. 5'-Monodeiodination of T4 was abolished by heating to 70 C for 30 min and was unaffected by EDTA. Propylthiouracil and methimazole did not inhibit T3 generation. Iopanoic acid, on the other hand, was a competitive inhibitor of the 5'-monodeiodination reaction, abolishing T3 production in a dose-dependent manner with a Ki of 3 microM. These data indicate that the bovine anterior pituitary contains significant T4 5'-deiodinase activity, which shares many properties of the type II 5'-deiodinase of the rat. Bovine anterior pituitary T4 5'-deiodinase appears to be predominantly localized in the rough endoplasmic reticulum.
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PMID:Subcellular localization of thyroxine 5'-deiodinase activity in bovine anterior pituitary. 406 44

Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b(5), and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, beta-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.
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PMID:Analytical study of microsomes and isolated subcellular membranes from rat liver. 3. Subfractionation of the microsomal fraction by isopycnic and differential centrifugation in density gradients. 415 Apr 90

Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20-30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2-3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.
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PMID:Isolation of a Golgi apparatus-rich fraction from rat liver. II. Enzymatic characterization and comparison with other cell fractions. 431 70

Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF(1), GF(2), GF(3)) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF(1) and GF(2), and along the outside of the cisternal membranes in GF(3). In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF(1) and within many VLDL-filled vacuoles in GF(1) and GF(2), indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF(3) and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF(1) and GF(2), and was not found in the cisternae in GF(3). The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF(1) and GF(3), representing primarily trans-Golgi elements from the secretory Golgi face, and GF(3) consisting largely of cis-Golgi components from the opposite face.
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PMID:Cytochemistry of Golgi fractions prepared from rat liver. 435 30

Homogenates of cultured rat embryo fibroblasts have been assayed for acid phosphatase, N-acetyl-beta-glucosaminidase, cathepsin D, acid deoxyribonuclease, cytochrome oxidase, NADH cytochrome c reductase, 5'-nucleotidase, inosine diphosphatase, acid pyrophosphatase, neutral pyrophosphatase, esterase, catalase, cholesterol, and RNA. The validity of the assay conditions was checked. Neutral pyrophosphatase is a readily soluble enzyme. Acid hydrolases, except acid pyrophosphatase, are particle-bound enzymes, which exhibit a high degree of structural latency. They are activated and solubilized in a parallel fashion by mechanical treatments and tensio-active agents. Catalase is also particle-bound and latent; activating conditions stronger than those for hydrolases are required to activate the enzyme. Acid pyrophosphatase, 5'-nucleotidase and inosine diphosphatase are firmly particle-bound, but not latent; they are not easily solubilized. In differential and isopycnic centrifugation, the latent hydrolases, cytochrome oxidase and catalase dissociate largely from each other; this suggests the occurrence of lysosomes and peroxisome-like structures besides mitochondria. The distribution patterns of 5'-nucleotidase and cholesterol are largely similar; digitonin influences their equilibrium density to the same extent; these two constituents are thought to be related to the plasma membrane. Inosine diphosphatase and acid pyrophosphatase are also partially associated with the plasma membrane, although some part of these enzymic activities probably belongs to other structures. NADH cytochrome c reductase is associated partly with the endoplasmic reticulum, partly with mitochondria.
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PMID:Analytical fractionation of homogenates from cultured rat embryo fibroblasts. 437 90

A procedure has been developed for the cytochemical localization of 5'-nucleotidase in isolated, unfixed, rat liver microsomes. Membranes were incubated with adenosine 5'-phosphate (5'-AMP) and Pb(NO(3))(2) and then isolated on sucrose density gradients: all the phosphate released was recovered with the membranes by this procedure. Adenosine 2'-phosphate (2'-AMP) and adenosine 3', 5'-cyclic phosphate (3',5'-AMP) were shown to be competitive inhibitors, but not substrates, for purified 5'-nucleotidase and were employed to determine the specificity of the cytochemical reaction. It was found that the incubation conditions for the cytochemical assay did not affect the specificity of 5'-nucleotidase. Microsomes incubated as controls with Pb(2+), or Pb(2+) and 2'-AMP or 3',5'-AMP were of the same density, although slightly denser than microsomes incubated without Pb(2+), and were unassociated with lead precipitate when examined by electron microscopy; microsomes incubated with Pb(2+) and 5'-AMP were much denser and were stained heterogeneously with lead phosphate when examined by electron microscopy. Precipitates formed artificially from Pb(2+) and inorganic phosphate did not resemble the reaction product. Microsomes were, therefore, separated on sucrose gradients and the subfractions were examined cytochemically. Lead precipitates were associated with the majority of rough-surfaced vesicles, and the reaction product was distributed heterogeneously in all fractions. Vesicles which stained like the membranes of the bile canaliculi in isolated plasma membranes were observed in the lightest subfraction. The reaction product was localized on the outside surface of the microsomal membranes, and was solubilized by low concentrations of ethylenediaminetetraacetic acid. It is concluded that 5'-nucleotidase is present in the endoplasmic reticulum and that the microsome fraction contains, in addition, vesicles derived from the plasma membrane.
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PMID:Cytochemical localization of 5'-nucleotidase in subcellular fractions isolated from rat liver. I. The origin of 5'-nucleotidase activity in microsomes. 500 18

Effects of the dopamine agonist 2-bromo-alpha-ergocryptine (bromocriptine) on plasma and pituitary PRL and enzyme activities in lactating and postlactating rats have been investigated. Lactating rats which had been suckling their young for 3 days were given a single sc injection of bromocriptine or solvent. The treated and control animals were divided into 2 further groups. One group (lactating rats) was permitted to suckle their pups for a further 12 or 24 h; the young were removed from the other group (postlactating rats). Homogenates were prepared from the anterior pituitaries and assayed for organelle marker enzyme activities. When 0.5-500 micrograms bromocriptine were administered to lactating rats for 24 h, pituitary PRL was increased by all doses, but only the 500-micrograms dose significantly reduced plasma PRL. Total protein was unchanged, lysosomal acid PRL proteolytic activity increased 8-fold, N-acetyl-beta-glucosaminidase and beta-glucuronidase (lysosomes) were unchanged, acid phosphatase (lysosomes and endoplasmic reticulum) was increased by three of four doses, 5'-nucleotidase and alkaline phosphatase (plasma membrane) were increased 4-fold, neutral-alpha-glucosidase (endoplasmic reticulum) and malate dehydrogenase (mitochondria) were unchanged, and catalase (peroxisomes) was significantly increased. Bromocriptine (500 micrograms) administration to lactating and postlactating rats for 12 and 24 h significantly decreased the pituitary DNA but not the total protein content of the pituitaries in all animals. The lysosomal acid PRL proteolytic activity and the lysosomal enzyme activities, N-acetyl-beta-glucosaminidase and beta-glucuronidase, were increased by suckling withdrawal alone. Acid PRL proteolytic activity was further increased (to 18-fold) by coadministration of bromocriptine, whereas the increase in the activities of the other lysosomal marker enzymes was blocked. Malate dehydrogenase activity (mitochondria) was also increased by litter removal and blocked by bromocriptine. The activity of the plasma membrane markers 5'-nucleotidase and alkaline phosphatase were increased by litter removal, and bromocriptine further increased both enzyme activities. The activity of neutral-alpha-glucosidase (endoplasmic reticulum) was unchanged by any treatment. The results demonstrate that bromocriptine produces significant changes in the activities of lysosomal marker enzymes, particularly acid PRL proteolytic activity, as well as marker enzymes of plasma membranes and other organelles in pituitaries of lactating and postlactating rats.
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PMID:Effects of bromocriptine on pituitary organelle marker enzyme activities in lactating and postlactating rats: selective activation of lysosomal prolactin proteolytic activity. 608 93


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