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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)

Arrhenius plots of 5'-nucleotidase activity in microsomes or plasma membranes from rat liver exhibited transitions at approximately 35 degrees C. The enzyme was purified from homogenates after solubilization in 2% Triton X-100 and 1% sodium deoxycholate. After the initial steps of the purification, the enzyme was recovered in membranes, as judged by both thin section and freeze-fracture electron microscopy, which contained sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. The purest fractions of 5'-nucleotidase were enriched approximate 3,000-fold, consisted of similar membranes, but only contained sphingomyelin. Thermal transitions were detected in Arrhenius plots of 5'-nucleotidase after detergent solubilization, in the membranes which contained the three phospholipids, but not in the purified fraction which contained only sphingomyelin; transitions were also detected after reassociation of the purified enzyme with microsomal or plasma membrane lipids and phosphatidylcholine but not with phosphatidylethanolamine. Phosphatidylcholines containing specific fatty acids all affected the energy of activation of 5'-nucleotidase, and the detergent Sarkosyl, which has been shown to dissociate phospholipids from 5'-nucleotidase (Evans, W. H., and Gurd, J. W. (1973) Biochem. J. 133, 189-199), caused a marked decrease in the stability of the enzyme to heating. Inhibition of 5'-nucleotidase by concanavalin A followed by reactivation with alpha-methyl-D-mannoside resulted in linear Arrhenius plots of 5'-nucleotidase activity in membrane fractions, and in lower transition temperatures for the detergent, solubilized enzyme. It is concluded that in situ, 5'-nucleotidase interacts with both sphingomyelin and phosphatidylcholine; the first apparently influences the stability of the enzyme and the second, the energy of activation. In addition, the lipid environment of the enzyme seems to be altered as a result of lectin binding.
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PMID:The effects of phospholipids on the properties of hepatic 5'-nucleotidase. 625 95

High voltage free flow electrophoresis has been applied to the separation of human platelet membranes. After short treatment with neuraminidase at the whole cell level, three membrane vesicle subpopulations have been isolated. Using a surface label (125I-labeled Lens culinaris lectin), the marker enzyme NADH-cytochrome c reductase, and lipid analysis, two of the fractions have been identified as of surface origin and the other consists of intracellular membrane elements. The distribution of adenylate cyclase, leucyl aminopeptidase, 5'-nucleotidase and Ca2+-ATPase has also been investigated, and their usefulness as markers for the different membrane fractions has been evaluated. All three fractions are vesicular but differ in size and character. Their phospholipid and cholesterol contents have been determined, and the cholesterol/phospholipid ratios of the two surface fractions are over twice that of the intracellular membrane, which also has a significantly lower microviscosity as determined by fluorescence polarization using diphenyl hexatriene. The polypeptide profiles from sodium dodecyl sulfate-polyacrylamide gel electrophoresis are particularly distinctive, with actin present in the two surface membrane fractions and absent from the intracellular membranes. Myosin, confirmed by its ATPase characteristics, is almost exclusively localized in one of the surface membrane fractions, and actin-binding protein is a prominent feature of the other.
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PMID:Characterization of human platelet surface and intracellular membranes isolated by free flow electrophoresis. 626 Jul 85

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess p-nitrophenyl phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5'-nucleotidase inhibitor alpha, beta-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5'-nucleotidase. An antibody against mouse liver 5'-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5'-nucleotidase and the necessity for distinguishing between true 5'-nucleotidase and non-specific phosphatase activity is discussed.
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PMID:Absence of 5'-nucleotidase in porcine polymorphonuclear leucocyte membranes. 628 71

A surface membrane fraction of high purity and good yield has been prepared from homogenates of rabbit peritoneal polymorphonuclear leucocytes, using a preliminary sorbitol density gradient sedimentation followed by preparative high voltage electrophoresis in a thin flowing buffer film. Enrichment values for the plasma membrane marker enzyme 5'-nucleotidase and 125I-labelled Lens culinaris lectin, after the latter had been applied at the whole cell level, were 18-fold and 6-fold, respectively. Contamination of the surface membrane fraction by other organelles was negligible and approximately 1 mg of surface membrane protein can be obtained from 2 . 10(9) leucocytes. A triacylglycerol-rich, protein-poor fraction that lacks any definable structure in electron microscopy separates discretely from the surface membrane vesicles during electrophoresis. It is considered that this may be a contaminant not previously recognized as present in membrane fractions prepared by more conventional procedures.
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PMID:Preparation of a highly purified surface membrane fraction from rabbit polymorphonuclear leucocytes by high-voltage free-flow electrophoresis. 630 25

Three techniques for the disruption/recovery of tegumental free-surface plasmalemma were compared by (i) morphological examination of carcasses and centrifugally-derived isolates, (ii) specific enrichment of bound surface tags (lectin) and of "marker" enzymes for membrane, and (iii) assessment of total protein and lectin recovered by each procedure. Procedures compared included the use of Triton X-100, freezing and thawing, and high ionic strength calcium. Triton X-100 consistently provided the greatest amounts of recovered surface membrane on a per worm basis, whereas calcium retained the highest amounts of alkaline p-nitrophenyl phosphatase, adenosine triphosphatase, and adenosine monophosphatase activity. Ultrastructural examination of membrane isolates and worm carcasses prepared by freezing and thawing indicated that significant amounts of parenchymal material contaminated the membrane fractions. Thus results based on the freeze-thaw technique can be difficult to interpret.
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PMID:Comparison of calcium, freeze-thaw, and Triton X-100 tegumental disruption/recovery techniques applied to Schistosoma mansoni. 631 92

The localization of the Pseudomonas aeruginosa lectins (PA-I and PA-II) was studied using methods of osmotic shock, freezing and thawing and spheroplast formation. Very slight release of the two lectins occurred when P. aeruginosa was exposed to magnesium-osmotic shock or was frozen and thawed. Under these conditions, release of the periplasmic 5'-nucleotidase occurred, whereas no release of cytoplasmic glucose-6-phosphate dehydrogenase activity was detected. Formation of spheroplasts from P. aeruginosa by gradual removal of the bacterial envelopes revealed low lectin activity in the treatment fluids. Osmotic shock treatment of the lysozyme treated mureinoplasts resulted in low release of glucose-6-phosphate dehydrogenase and the two lectins (10-13%) and a considerable activity (38.4%) of 5'-nucleotidase. The presence of the lectins on the outer and the cytoplasmic membranes enabled intact cells and spheroplasts of P. aeruginosa to agglutinate papain-treated human erythrocytes. These results indicate that the two lectins are located mainly in the cytoplasm with small fractions on the cytoplasmic and outer membranes and in the periplasmic space.
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PMID:The intracellular localization of Pseudomonas aeruginosa lectins. 631 95

Plasma membrane vesicles purified from pig mesenteric lymph nodes were solubilized using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide, and lentil lectin receptor glycoproteins were isolated by affinity chromatography. The receptor fraction showed 12 major bands on SDS-polyacrylamide gel electrophoresis representing 8-9% of the membrane protein. 5'-Nucleotidase (EC 3.1.3.5) was very effectively solubilized by the detergent and was recovered in high yield in the receptor fraction. Receptor glycoproteins were reassembled into large unilamellar vesicles of phosphatidylcholine/phosphatidylserine (mean diameter 0.235 microm) using a detergent dialysis technique. Sixty to seventy percent of the glycoprotein and most of the 5'-nucleotidase activity is associated with the phospholipid vesicles. 5'-Nucleotidase is reassembled in a symmetrical fashion and is inhibited by binding of concanavalin A, lentil lectin and pea lectin but not by succinyl-concanavalin A. Measured values for Ki and maximal inhibition are similar to those observed with intact plasma membrane vesicles. Hemagglutination inhibition studies showed that the reassembled receptors effectively bind lentil lectin. Thus lymphocyte membrane glycoproteins reassembled into phospholipid vesicles seem to retain at least part of their function in that enzyme activities such as 5'-nucleotidase remain intact and the receptors effectively bind lentil lectin.
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PMID:Functional reassembly of lymphocyte lentil lectin receptor glycoproteins into lipid bilayer vesicles. 683 Jul 99

Plasma membranes have been prepared from purified pachytene spermatocytes, round spermatids and residual bodies of the adult mouse testis using procedures modified from other authors'. Isolated membranes have been examined using electron microscopy, lectin binding and enzymic assays. Ultrastructural observation reveals smooth unit-membrane vesicles from 0.4-1.7 micrometer diameter. No contamination by nuclei, mitochondria or lysosomes is detected microscopically. Radiolabelled lectin-binding experiments [125I-RCAI, 125I-green pea lectin] indicate that cell surface label cofractionates with material identified morphologically as plasma membrane. Estimates of total recovery of membrane, based upn the lectin data, average 33%. Biochemical analysis of subcellular markers reveal that no detectable DNA and only 1.2% of the total cellular RNA cofractionate with membranes. A variety of enzyme assays suggests little contamination by cytosol enzymes, Golgi material or mitochondria. Assays of 5'-nucleotidase (E.C. 3.1.3.5) indicate that this enzyme is not a major component of developing mouse spermatogenic cell membranes. Instead, Sertoli cells represent the most important source of this enzyme in the adult seminiferous tubule. Polyacrylamide gel analysis of membranes isolated from purified germ cells reveals significant differences in the protein compositions of pachytene spermatocyte and round spermatid membranes. The preparation of highly purified plasma membranes from homogeneous populations of spermatogenic cells should facilitate the biochemical characterization of cell surface antigens specific to developing male germ cells.
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PMID:Isolation of plasma membranes from purified mouse spermatogenic cells. 741 22

Twenty-four intervertebral discs from the lumbar region of postmortem human subjects were investigated by immunohistochemical and histochemical methods to evaluate the pattern of blood and lymph vessels of the discus intervertebralis and surrounding tissue at different ages. Antibodies against the basement membrane component laminin, and the lectin Ulex europaeus agglutinin as a marker for L-fucose in endothelial cells, were used to make the blood vessels visible. The 5'-nucleotidase activity in lymph endothelium served as a marker for lymphatic vessels. The dorsolateral parts of the connective tissue adjacent to the discus intervertebralis were well vascularized in all age groups examined. Vessels in the outer anulus fibrosus were detected in young individuals up to 20 years of age. Vascular canals, i.e. blood vessels in the cartilage end plate, were seen up to 7 years of age. Lymph capillaries are first described here penetrating the disc and peridiscal tissue and accompanying most of the small blood vessels into the areas specified.
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PMID:Detection of lymph and blood vessels in the human intervertebral disc by histochemical and immunohistochemical methods. 833 22

Immunohistochemical (antibodies against laminin) and histochemical methods (Ulex europaeus lectin, 5'-nucleotidase activity) were used to describe the vascular pattern of human intervertebral discs and the surrounding tissue at different ages. Blood and lymph vessels were found in the connective tissue outside the annulus in all age groups. In the annulus blood vessels and lymphatics were detected up to 20 years of age, in the cartilage end-plate blood vessels appeared up to 7 years of age (cartilage canals). In the nucleus pulposus neither blood nor lymph vessels could be seen at any age. The occurrence of blood and lymph vessels in growing intervertebral discs help us to understand childhood discitis without simultaneous affection of the vertebral body.
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PMID:Lymph and blood supply of the human intervertebral disc. Cadaver study of correlations to discitis. 845 43


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