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)

We have developed a method for isolation of plasma membranes from rabbit endometrium, with high yield and purification. Endometrial homogenates are precipitated with calcium chloride and the resulting supernatant is fractionated by centrifugation in a self-forming gradient of 20% Percoll. Before fractionation, the intact luminal epithelial surface was labelled with 125I-labelled soyabean agglutinin. Between buoyant densities of 1.015 and 1.017 g/ml, a discrete peak of surface label was obtained, which coincided with activities for 5'-nucleotidase and alkaline phosphatase, enzyme markers for the plasma membrane. This peak was well separated from the majority of cellular protein, and from marker enzyme activities for mitochondria and microsomes (NADH cytochrome C reductase) and lysosomes (acid phosphatase). Electron microscopy of the purified membranes showed membrane sheets and vesicles free from other cellular organelles. Analysis of detergent-soluble membrane proteins, fractionated by concanavalin A-affinity chromatography, revealed differences in the protein pattern of membranes from uteri of rabbits receptive (Day 6 of pregnancy) and non-receptive (Day 3) for implantation. The method will be useful for generation of immunological and affinity probes for surface antigens involved in ovoimplantation.
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PMID:Purification of rabbit endometrial plasma membranes from receptive and non-receptive uteri. 299 83

The kinetic properties of highly purified human placental cytoplasmic 5'-nucleotidase were investigated. Initial velocity studies gave Michaelis constants for AMP, IMP, and CMP of 18, 30, and 2.2 microM, respectively. The enzyme shows the following relative Vmax values: CMP greater than UMP greater than dUMP greater than GMP greater than AMP greater than dCMP greater than IMP. The activity was magnesium-dependent, and this cation binds sequentially with a Km of 14 microM for AMP and an apparent Km of 6 mM for magnesium. A large variety of purine, pyrimidine, and pyridine compounds exert an inhibitory effect on enzyme activity. IMP, GMP, and NADH produce almost 100% inhibition at 1.0 mM. Nucleoside di- and triphosphates are potent inhibitors. ATP and ADP are competitive inhibitors with respect to AMP and IMP as substrates with Ki values of 100 and 15 microM, respectively. Inorganic phosphate is a noncompetitive inhibitor with Ki values of 19 and 43 mM. Nucleosides and other compounds studied produce only a modest decrease of enzyme activity at 1 mM. Our findings suggest that the enzyme is regulated under physiological conditions by the concentrations of magnesium, nucleoside 5'-monophosphates, and nucleoside di- and triphosphates. The nucleotide pool concentration regulates the enzyme possibly by a mechanism of heterogeneous metabolic pool inhibition. These properties of human placental cytoplasmic 5'-nucleotidase may be related to the control of nucleotide degradation in vivo.
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PMID:Human placental cytoplasmic 5'-nucleotidase. Kinetic properties and inhibition. 300 Oct 58

An accelerated method is developed for isolating a fraction of plasma membranes of pig myometrium using ultracentrifugation within the sucrose density gradient (15% and 30%). The membranes possessed the high activity of 5'-nucleotidase and Na+, K+-ATPase and the low activity of rhotenon-insensitive NADH-cytochrome c reductase. The vesicularized preparations of plasma membranes are able of ATP-dependent accumulation of Ca2+ (7.5 +/- 0.3 nmol. 45Ca2+ per 1 mg of protein for 15 min). Phosphate increases the calcium accumulation in the presence of ATP and Mg2+. Ionophore A 23187 promotes a complete and rapid release of the previously active-accumulated calcium. The release of 45Ca2+ accumulated by the membrane fraction may be reached by introduction of 1 mM EGTA or DS-Na into the incubation medium, that evidences for the cation accumulation inside closed structures. Using concanavalin-A-sepharose 4B it is shown that 60% of membrane vesicles are turned inside out. The low saponine concentrations (0.0005%) which inhibit Ca2+-accumulation by plasma membranes but not by the endoplasmic reticulum inhibit this process by 60-70% in preparations of the isolated membrane fraction. The method has certain advantages over the previously applied methods used for isolating of plasma membrane fragments from smooth muscles.
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PMID:[Isolation and characteristics of the plasma membrane fraction from the swine myometrium]. 301 62

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

Muscle biopsies from six horses with clinical histories of muscle atrophy, muscle tremors, myopathic symptoms, unsteadiness of pelvic limbs and progressive ataxia were examined. Muscle biopsies were studied with enzyme histochemical techniques to evaluate the diagnostic values of these methods in cases suspected of suffering from neuromuscular disorders. Hypertrophy, atrophy, fibre splitting, waxy degeneration, phagocytosis and necrosis were seen in haematoxylin eosin stained sections of the different cases. Fibre type predominance and fibre type grouping were seen in the calcium ion stimulated myosine ATP-ase (Ca-ATP-ase) stained sections of some cases. 'Moth-eaten fibres' were demonstrated in three cases by staining with NADH: nitro blue tetrazolium oxidoreductase (NADH-TR), succinate dehydrogenase (SDH), NADH dependent malate dehydrogenase, cytochrome c oxidase and by lactate dehydrogenase. The catabolic enzymes, acid phosphatase (ACP) and 5'-nucleotidase were active in cases with fibre phagocytosis. The oxidative part of the pentose phosphate pathway in myopathic tissue seemed to be important in three cases, demonstrated by the increased activity of glucose-6-phosphate dehydrogenase (GPDH) and 6-phosphogluconate dehydrogenase (PGDH). The important feature of diseased horse muscle was that the pathohistochemical changes were exactly the same as in diseased skeletal muscles of humans. The application of tissue saving enzyme histochemical techniques can be recommended in the study of muscle tissue from horses suffering from suspected neuromuscular disorders.
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PMID:Enzyme histochemistry on muscle biopsies as an aid in the diagnosis of diseases of the equine neuromuscular system: a study of six cases. 336 6

Subcellular fractions of nuclei, mitochondria, endoplasmic reticulum, plasma membrane and cytosol were prepared from liver and hepatoma 7288CTC. Marker enzyme activities, biochemical compositions and electron microscopy were used to establish purity. Hepatoma NADH: cytochrome C reductase and 5'-nucleotidase exhibited abnormal subcellular distributions. The lipids from the subcellular fractions were examined in detail. Mitochondria and plasma membranes were characterized by elevated percentages of diphosphatidylglycerol and sphingomyelin, respectively, in both tissues. All hepatoma subcellular fractions contained dramatically elevated levels of sphingomyelin and cholesterol, two components that form preferential strong complexes in vitro. The fatty acid composition of hepatoma sphingomyelin differed markedly from liver and, unlike liver, did not exhibit organelle specific compositions. Some hepatoma lipid classes contained reduced percentages of palmitate while others contained higher levels. Hepatoma phosphatidylcholine and phosphatidylethanolamine from organelles contained lower percentages of long chain polyunsaturated fatty acids than liver. Generally, unique fatty acid profiles exhibited by individual phospholipid classes of liver subcellular fractions were absent or much reduced in the hepatoma. The ratios of oleate to vaccenate were near one for most of the phospholipid classes of most liver fractions, but all hepatoma classes, with few exceptions, contained a much higher percentage of oleate in all subcellular fractions. The hypothesis is proposed that the origin of some acyl moieties for the biosynthesis of various hepatoma lipid classes differs from liver sources. The possible changes in acyl pools, sources and compartments for complex lipid biosynthesis could result in change in the quantities of molecular species that could contribute to the abnormal properties of the hepatoma membranes.
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PMID:A comparison of lipids from liver and hepatoma subcellular membranes. 371 48

The series introduced by this paper reports the results of a detailed analysis of the microsomal fraction from rat liver by density gradient centrifugation. The biochemical methods used throughout this work for the determination of monoamine oxidase, NADH cytochrome c reductase, NADPH cytochrome c reductase, cytochrome oxidase, catalase, aminopyrine demethylase, cytochromes b(5) and P 450, glucuronyltransferase, galactosyltransferase, esterase, alkaline and acid phosphatases, 5'-nucleotidase, glucose 6-phosphatase, alkaline phosphodiesterase I, N-acetyl-beta-glucosaminidase, beta-glucuronidase, nucleoside diphosphatase, aldolase, fumarase, glutamine synthetase, protein, phospholipid, cholesterol, and RNA are described and justified when necessary.
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PMID:Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods. 415 Apr 88

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

Zonal centrifugation has been used to isolate a fraction from bovine liver which appears to be derived from the Golgi apparatus. Morphologically, the fraction consists mainly of sacs and tubular elements. Spherical inclusions, probably lipoproteins, are occasionally seen in negative stains of this material. The preparation is biochemically unique. UDP-galactose:N-acetyl glucosamine, galactosyl transferase activity is concentrated about 40-fold in this fraction compared to the homogenate. Rotenone- or antimycin-insensitive DPNH- or TPNH- cytochrome c reductase activities are 60-80% of the level of activities found in microsomes. Purified organelles from bovine liver such as plasma membranes, rough microsomes, mitochondria and nuclei have negligible levels of galactosyl transferase. Some activity is present in smooth microsomes but at a level compatible with the possible presence of Golgi membranes in this fraction. The Golgi fraction does not contain appreciable amounts of enzymes such as ATPase, 5'-nucleotidase, glycosidase, glucose-6-phosphatase, acid phosphatase, or succinate-cytochrome c reductase. Similar fractions isolated from bovine epididymis also have very high levels of galactosyl transferase. The fraction is heavily osmicated when incubated for long periods of time at elevated temperatures, a characteristic property of Golgi membranes.
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PMID:Isolation and characterization of Golgi membranes from bovine liver. 424 7

Plasma membranes from KB cells were isolated by the method of latex bead ingestion and were compared with those obtained by the ZnCl(2) method. Optimal conditions for bead uptake and the isolation procedure employing discontinuous sucrose gradient centrifugation are described. All steps of preparative procedure were monitored by electron microscopy and specific enzyme activities. The plasma membrane fraction obtained by both methods is characterized by the presence of the Na(+) + K(+)-activated ATPase and 5'-nucleotidase, and contains NADPH-cytochrome c reductase and cytochrome b(5). The latter two enzymes are also present in lower concentrations in the microsomal fraction. Unlike microsomes which are devoid of the Na(+) + K(+)-activated ATPase and which contain only traces of 5'-nucleotidase activity, the plasma membrane fraction contains only trace amounts of the rotenone-insensitive NADH-cytochrome c reductase but no cytochrome P-450, both of which are mainly microsomal components. Morphologically the plasma membrane fraction isolated by the latex bead method is composed of vesicles of 0.1-0.3 microm in diameter. On the basis of the biochemical and morphological criteria presented, it is concluded that the plasma membrane fraction isolated by the above methods are of high degree of purity.
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PMID:Isolation and properties of the plasma membrane of KB cells. 428 30


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