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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)

The subcellular localization of adenylate cyclase (ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1) in bovine corpus luteum was studied using isotonic and hypotonic homogenization and fractionation conditions. All fractions prepared were assayed for adenylate cyclase, marker enzymes and DNA. Only plasma membrane marker enzyme, 5'-nucleotidase paralleled the distribution of adenylate cyclase under both isotonic and hypotonic conditions (conditionsoth isotonic and hypotonic conditions (coefficient of correlation = 0.95). Two main fractions prepared under hypotonic conditions were subfractionated by discontinuous sucrose gradient centrifugation. The highest amount of adenylate cyclase was found in a fraction having a density approximately equal to 1.13 g/cm3. The specific activity of this fraction was 4--6 times higher than that of the homogenate. The electron microscopic study of this fraction revealed the presence of a single type of particulate material consisting of small vesicles exhibiting a typical unit membrane structure. It is concluded that this adenylate cyclase is primarily localized in the plasma membranes. Basal adenylate cyclase activity of plasma membranes was stimulated 2--3 times by luteinizing hormone (10 mug/ml), 3--4 times by prostaglandin E2 (10 mug/ml), 4--6 times by NaF (0.01 M) and two times by methanol (0.2%).
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PMID:Subcellular localization and partial characterization of bovine corpus luteum adenylate cyclase. 114 60

1. Human tumour KB cells growing in suspension culture were labelled by lactoperoxidase-catalysed iodination. Several major radioactively labelled proteins were detected by poly-acrylamide-gel electrophoresis in sodium dodecyl sulphate. 2. After reduction with 2-mercaptoethanol the major radioactive electrophoretic bands migrated as substances with apparent molecular weights of about 90,000, 70,000, 60,000, 50,000 and 34,000 and corresponded closely to the positions at which the major glycosylated polypeptide subunits of KB-cell homogenates migrated during electrophoresis under the same conditions. 3. All the iodinated protein bands except one were present in purified preparations of KB plasma membranes. 4. Most of the 50,000-molecular-weight species, supposedly a surface protein component labelled during iodination of intact and viable KB cells by a non-penetrating enzyme reagent, appeared in a crude nuclear pellet during fractionation. 5. The glyco-protein nature of the major external iodinated species of KB cells was confirmed by adsorption chromatography of these substances, dissolved in low concentrations of Triton X-100, on a lectin-Sepharose column. Two major enzyme markers of the KB plasma membrane, 5'-nucleotidase and alkaline phosphatase were also found to be glycoproteins. 6. Enzyme-catalysed incorporation of radioactive iodine into a fraction of low molecular weight and soluble in chloroform-methanol mixtures also occurred during lactoperoxidase treatment of intact KB cells. The partial characterization of this fraction is briefly described.
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PMID:Surface labelling for human tumour KB cells. Iodination and fractionation of membrane glycoproteins. 120 Oct 9

We report a rapid and reproducible assay for activity of human erythrocyte pyrimidine 5'-nucleotidase and deoxypyrimidine 5'-nucleotidase. The nucleotides CMP, UMP, dUMP, dCMP or dTMP are individually incubated 30 min at 37 degrees C with erythrocyte hemolysate and 4 mM magnesium chloride in Tris, pH 7.5. Data are provided for standardization of the reaction with each substrate. Individual nucleoside products are assayed in less than 10 min by reversed-phase high-performance liquid chromatography at 280 nm with 0-14% methanol in 0.01 M potassium dihydrogen phosphate. This is the first report of a high-performance liquid chromatographic assay system which allows quantitation of the activity of pyrimidine 5'-nucleotidase isozymes using five individual pyrimidine and deoxypyrimidine nucleotides as the substrates.
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PMID:Assay of human erythrocyte pyrimidine and deoxypyrimidine 5'-nucleotidase by isocratic reversed-phase high-performance liquid chromatography. 298 6

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

A number of acyclo nucleosides of benzimidazole derivatives has been synthesized, in which the benzimidazole ring includes substituents at C(5), C(6) and C(2). The acyclic chains which replace the sugar moiety are 2',3'-dihydroxypropyl, 2'-hydroxyethoxymethyl and 1',5'-dihydroxy-4'-hydroxymethyl-3'- oxypentyl -2' (R), each of which corresponds to some fragment of the ribose ring. 1H NMR spectroscopy has been employed to determine the conformations of these acyclic chains in solutions of fully deuterated dimethylsulfoxide and methanol, utilizing for this purpose vicinal proton-proton coupling constants, and the new Karplus relation developed by Haasnoot , de Leeuw & Altona ( Tetrahedron , 36, 2783-2792, 1980). The data thus obtained are compared with those available for the solid state from X-ray diffraction data, and should be applicable to other classes of acyclonucleosides . Nucleotides of the three types of acyclo benzimidazole nucleosides have also been prepared, and their susceptibilities to snake venom 5'-nucleotidase examined. In contrast to acycloG , the nucleoside analogues did not exhibit significant in vitro activity against herpes simplex virus type 1 or influenza virus.
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PMID:Acyclonucleosides: acyclobenzimidazole nucleoside and nucleotide analogues and conformations of the acyclic chains by means of NMR spectroscopy. 632 39

Among the biological exposure indices of lead, lead in plasma was the most direct indicator of current exposure. Lead mobilized into plasma as well as in urine could be used as an indicator of the internal dose of lead. The ratio of non-treated to restored activity of delta-aminolevulinic acid dehydratase (ALA-D) was a more specific index than ALA-D activity itself at low levels of lead exposure, excluding the familial or genetic variation in the activity. The methods using HPLC for determining heme intermediate improved the evaluation of the lead effect: delta-aminolevulinic acid in plasma, blood, and urine (ALA-P, ALA-B, and ALA-U), coproporphyrin in urine, and zinc protoporphyrin in blood (ZP). ROC (Receiver operating characteristic) curve analyses indicated that the diagnostic values for lead exposure decreased in the order ALA-D ratio > ALA-D activity = ALA-P > ALA-U = ZP. Pyrimidine 5'-nucleotidase activity or pyrimidine nucleotide concentrations in blood was also useful for the monitoring or diagnosis of lead intoxication. Using the HPLC method with inclusion compounds in the mobile phase, hippuric acid, methylhippuric acids, mandelic acid and phenylglyoxylic acid could be simultaneously determined in the urine of workers exposed to a mixture of toluene, xylenes, and ethylbenzene. The correction of the urinary metabolite concentration for specific gravity or creatinine allowed the more specific evaluation of the solvent exposure. In the biological monitoring of chlorinated hydrocarbons such as trichloroethylene, prolonged excretion of the metabolites resulted in a bias between metabolite concentrations and TWA levels of the solvent in a day. The background levels of 2,5-hexanedione (HD) were affected by acid hydrolysis conditions, age, sex and lipid metabolism. Substances hydrolyzed to HD in urine from non-exposed subjects were different from HD detected in the workers exposed to n-hexane. Urinary concentrations of N-acetyl-S-(N-methylcarbamoyl) cysteine (AMCC) served as an index of the average exposure to N, N-dimethylformamide during several preceding work days and may indicate the internal dose, while N-methylformamide may be an index of daily exposure. A simple and rapid method for the determination of urinary alkoxyacetic acids was recently developed for the biological monitoring of workers exposed to glycolethers and their acetates. Urinary butoxy acetic acid (free plus conjugated ones) could be simply determined by gaschromatography after acid hydrolysis of urine. The urinary acetone or methanol concentration determined by the head space technique was also useful for the biological monitoring of workers exposed to isopropanol and/or acetone, or methanol, respectively. Evaluation of exposure to the solvents described above could be carried out by comparing the urinary metabolite concentrations with reference values and the biological exposure index values which were defined as the urinary metabolite concentration corresponding to the threshold value for each solvent.
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PMID:[Studies on the evaluation of exposure to industrial chemicals]. 868 99

The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed. 1592U89 was anabolized to its 5'-monophosphate (MP) by the recently characterized enzyme adenosine phosphotransferase, but neither its diphosphate (DP) nor its TP was detected. The MP, DP, and TP of CBV were found in cells incubated with either 1592U89 or CBV, with CBV-TP being the major phosphorylated species. We confirmed that CBV is phosphorylated by 5'-nucleotidase and that mycophenolic acid increased the formation of CBV-TP from CBV 75-fold. However, mycophenolic acid did not stimulate 1592U89 anabolism to CBV-TP. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not inhibit CBV-TP formation from CBV or 1592U89, whereas the adenylate deaminase inhibitor 2'-deoxycoformycin selectively inhibited 1592U89 anabolism to CBV-TP and reversed the antiviral activity of 1592U89. 1592U89-MP was not a substrate for adenylate deaminase but was a substrate for a distinct cytosolic deaminase that was inhibited by 2'-deoxycoformycin-5'-MP. Thus, 1592U89 is phosphorylated by adenosine phosphotransferase to 1592U89-MP, which is converted by a novel cytosolic enzyme to CBV-MP. CBV-MP is then further phosphorylated to CBV-TP by cellular kinases. This unique activation pathway enables 1592U89 to overcome the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity.
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PMID:Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89. 914 76

An isocratic HPLC method for the simple and selective determination of adenine nucleoside and nucleotides has been developed. The separation is achieved at room temperature by reversed-phase chromatography (Shiseido, Capcell Pak C18). A mixture of 0.1 M triethylamine (TEA) phosphate buffer and methanol (95:5, v/v) is used as a standard eluent. Influence of pH and concentrations of organic modifiers and TEA ion on capacity factors of adenine compounds has been investigated. It has been also found that the TEA ion in the eluent is adsorbed onto the reversed-phase surface. The results clearly demonstrate that ion-pair formation with TEA ion occurs probably both in the mobile phase and on the stationary phase and governs the retention of adenine and nucleotides in the present system. The HPLC system is applied to the analysis of adenine nucleotides formed as intermediates in the synthesis of 3'-phosphoadenosine 5'-phosphosulphate (PAPS) and to the assays of ATPases and 5'-nucleotidase activities in rat liver plasma membrane. This method is a new type of ion-pair reversed-phase HPLC system and is suitable for the separation of highly polar organic anions, especially for adenine nucleotides.
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PMID:Ion-pair reversed-phase high-performance liquid chromatography of adenine nucleotides and nucleoside using triethylamine as a counterion. 944 63

A group of rats were administered a methanolic extract of Eupatorium adenophorum (Ageratina adenophora) oven-dried (60 degrees C) leaf powder and a partially purified fraction from the methanolic extract. Administration of the methanolic extract and the partially purified fraction elicited a significant increase in total and conjugated bilirubin, alkaline phosphatase, 5'-nucleotidase and transaminases. Histopathology of the livers from these animals revealed dilated bile ducts and proliferative changes. Hepatocytes around the bile ducts showed necrotic changes. Biochemical and histopathological changes resembled those observed in response to administration of whole leaf powder. The hepatotoxin present in E. adenophorum leaves can be extracted with methanol and partially purified further using the procedure described.
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PMID:Hepatotoxicity in rat induced by partially purified toxins from Eupatorium adenophorum (Ageratina adenophora). 1107 39

In this study we have revised our original procedure of yeast metabolites extraction. We showed that: (a) less than 5% of intracellular metabolites leaks out during the step of rapid arrest of cellular metabolism by quenching yeast cells into a 60% methanol solution kept at -40 degrees C; and (b) with a few exception, the stability of metabolites were not altered during the 3 min boiling procedure in a buffered ethanol solution. However, there was a loss of external added metabolites of 5-30%, depending on the type of metabolites. This was mainly attributable to their retention on cellular debris after ethanol treatment, which prevented centrifugation of the cellular extracts before evaporation of ethanol. We further simplified our previous high-performance ionic chromatography (HPIC) techniques for easier, more reliable and robust quantitative measurements of organic acids, sugar phosphates and sugar nucleotides, and extended these techniques to purine and pyrimidine bases, using a variable wavelength detector set at 220 and 260 nm in tandem with a pulsed electrochemical or suppressed conductivity detector. These protocols were successfully applied to a glucose pulse to carbon-limited yeast cultures on purines metabolism. This study showed that glucose induced a fast activation of the purine salvage pathway, as indicated by a transient drop of ATP and ADP with a concomitant rise of IMP and inosine. This metabolic perturbation was accompanied by a rapid increase in the activity of the ISN1-encoded specific IMP-5'-nucleotidase. The mechanism of this activation remains to be determined.
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PMID:Revised procedures for yeast metabolites extraction: application to a glucose pulse to carbon-limited yeast cultures, which reveals a transient activation of the purine salvage pathway. 1719 50


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