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 perfused isolated rat livers with hypocalcemic (4.4 mg 100 ml) Krebs-Ringer bicarbonate albumin buffer. After 15 min of perfusion, a substance appeared in the perfusate which decreased rat renal adenylate cyclase activation by parathyroid hormone (PTH). The material in the perfusate was purified greater than 50,000-fold by Bio-Gel P-10 chromatography. The purified antagonist decreased the activation of rat renal cortical adenylate cyclase by PTH, glucagon, and epinephrine 75 to 100%. Concentration response curves for each of the hormones indicated a noncompetitive interaction of the inhibitor with the hormone. The inhibition was not species-specific, as the activation of the parathyroid hormone-responsive adenylate cyclase in cat renal cortex was also abolished by the inhibitor from the perfused rat liver. The inhibitor is a peptide, Mr equal to similar to 1000, which is heat-stable, acid-stable, alkai-labile, and is destroyed by trypsin, leucine aminopeptidase, and elastase. It is not destroyed by phosphodiesterase, 5'-nucleotidase, alkaline phosphatase, neuraminidase, RNase, or phospholipase A. The inhibitor is not produced by isolated rat livers perfused with normocalcemic perfusion media. It is unclear whether the peptide is synthesized by the liver or whether it is a breakdown product of a larger peptide or protein in the liver. This is the first reported peptide inhibitor of adenylate cyclase.
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PMID:Isolation of a unique peptide inhibitor of hormone-responsive adenylate cyclase. 16 24

A Golgi apparatus-rich fraction and a plasma membrane-rich fraction were isolated from a common homogenate of rat liver. Their respective buovant densities, appearances in the electron microscope and 5'-nucleotidase and UDP-galactose ovalbumin galactosyltransferase activities were in accord with published data on separately isolated Golgi apparatus-rich and plasma membrane-rich fractions. Contamination by endoplasmic reticulum and mitochondria was low. Gel electrophoresis of the membrane proteins of the Golgi apparatus-rich and plasma membrane-rich fractions (separately and mixed) showed a close similarity. After Neville's demonstration that electrophoretic patterns of membrane protein subunits from different subcellular fractions are easily distinguishable, the present work demonstrates an unusually close relationship between the Golgi apparatus membrane and the cell membrane. It is possible that membrane similarity may be mediated by the transfer of membrane-bound vesicles from the Golgi apparatus to the cell membrane.
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PMID:Similarities of the Golgi apparatus membrane and the plasma membrane in rat liver cells. 17 74

The human neutrophil neutral peptide-generating protease was associated with the plasma membrane marker 5'-nucleotidase on sucrose density gradient centrifugation of sonicates of granule-free fractions following homogenization and velocity sedimentation. The two activities were also associated on sucrose density gradient fractionation of plasma membranes obtained by hypotonic lysis in EDTA containing buffers, a technique which minimizes aggregation. Treatment of fractions containing these enzymatic activities with 1-0 M NaCl separated the neutral peptide-generating proteasein to the eluate while leaving the 5'-nucleotidase in the pellet. Gel filtration of the solubilized neutral peptide-generating protease through Sephadex G-100 in 1-0 M NaCl demonstrated that the protease had an approximate mol. wt of 20,000 while filtration in physiological salt concentrations yielded activity only in the excluded volume. In both cases, there was complete recovery of neutral peptide-generating activity suggesting that the filtration characteristics of the protease were determined by the salt concentration. The solubilized purified protease, the whole cell sonicates, and the intact cells interacted with heat-inactivated plasma to yield the same product, a neutral peptide with a 1000 molecular weight and an isoelectric point of 7-2-7-6. The neutral peptide-generating protease in each instance was inhibited in dose-response fashion by alpha-1-antitrypsin, LBTI, and DFP. Only 30-60% of the protease sites were functional on intact cells as revealed by substrate cleavage or were available to inhibitors. The neutrophil protease which generates neutral peptide is an extrinsic plasma membrane protein with an approximate mol. wt of 20,000 which functions as an ectoenzyme.
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PMID:A neutrophil-dependent pathway for the generation of a neutral peptide mediator. II. Subcellular localization of the neutrophil protease. 87 75

5'-Nucleotidase (EC 3.1.3.5) is widely distributed in nature. However, it could not be detected in rat liver, because of the presence of specific inhibitors. Such inhibitors were also found in other tissues of rat, but at lower concentrations than that in the liver. The inhibitor activity was enriched in the membrane fraction and was also present in the cytosol fraction. It was sensitive to treatment with 6M urea and trypsin, while heating in a boiling water bath for 10 min or dialysis reduced the activity only slightly. Gel filtration or Sephadex G-50 yielded two types of inhibitors. Inhibitor I inhibited brain 5'-nucleotidase while inhibitor II inhibited both the brain and liver enzymes. Inhibitor II on further purification on CM Sephadex C-25 yielded five fractions with inhibitor activity of which inhibitor IIC was electrophoretically homogeneous. It had a molecular weight of 8500 by SDS gel electrophoresis, was rich in basic amino acids and had a high proportion of beta structure. Interaction of the inhibitor with 5'-nucleotidase brought about modifications in the secondary structure of the inhibitor as seen from the circular dichroism spectrum.
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PMID:Isolation and characterization of 5'-nucleotidase inhibitor from rat liver. 139 14

Phosphatidate phosphohydrolase (PAP)-catalysed dephosphorylation of phosphatidic acid to diacylglycerol is an important step in glycerolipid metabolism and cell-signalling. Gel filtration chromatography on Superose 6 and anion-exchange chromatography on Mono Q of rat liver subcellular fractions has provided physical evidence for the presence of two distinct forms of PAP activity. One form was sensitive to inhibition by N-ethylmaleimide (NEM), had an apparent M(r) of 540,000 and was eluted from the anion-exchange column by 0.35 M NaCl, while the other was insensitive to inhibition by NEM, had an apparent M(r) of 240,000 and was eluted from the anion-exchange column by 0.15 M NaCl. Studies on the subcellular distribution of these two enzymes, using 5'-nucleotidase as a plasma membrane marker, demonstrated that the NEM-sensitive form was predominantly cytosolic but translocated to the microsomal membranes in response to oleate. The NEM-insensitive form was predominantly located in the plasma membrane but a small proportion (approx. 10%) of total cell activity was present on the endoplasmic reticulum. The implications of these results for the likely roles of the two different forms of PAP in fatty acid esterification and cell-signalling are discussed.
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PMID:Physical evidence for the presence of two forms of phosphatidate phosphohydrolase in rat liver. 162 38

5'-Nucleotidase is a member of a recently identified class of membrane proteins that is anchored via a phosphatidylinositol-containing glycolipid. The enzyme was readily solubilized with full retention of catalytic activity by nonionic and anionic detergents such as alkylthioglucosides, deoxycholate, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS), while the cationic detergent dodecyltrimethylammonium bromide (DTAB) caused loss of activity. 5'-Nucleotidase was released only at high detergent concentrations, suggesting that it is tightly associated with the membrane. DTAB and deoxycholate caused a loss of heat stability, while alkylthioglucosides had no effect. CHAPS produced a remarkable increase in the heat stability of the partially purified (glycoprotein fraction) and purified enzyme. Arrhenius plots of solubilized 5'-nucleotidase showed "break points" for all detergents in the temperature range 30-37 degrees C. SDS-PAGE of pure 5'-nucleotidase showed a single subunit of molecular mass 70 kilodaltons (kDa), while sucrose density gradient sedimentation gave a peak of activity corresponding to 132 kDa, indicating that the enzyme exists as a dimer. Gel filtration of the solubilized enzyme in several detergents showed apparent molecular masses between 200-630 kDa, suggesting that lymphocyte 5'-nucleotidase may be present in high molecular mass aggregates in its native state.
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PMID:Solubilization, characterization, and detergent interactions of lymphocyte 5'-nucleotidase. 255 35

The inhibition of the cell surface enzyme 5'-nucleotidase by concanavalin A is being studied as a model for understanding transmembrane modulation of cell surface functions. Nucleotidase of 13762 MAT-C1 ascites rat mammary adenocarcinoma cells is inhibited by concanavalin A in a noncooperative process. When cells are treated with the cytoplasmic effectors cytochalasins, colchicine, energy poisons, calcium plus ionophore or hypotonic buffers, the concanavalin A inhibition of the enzyme becomes cooperative. 5'-Nucleotidase of isolated MAT-C1 microvilli is also inhibited by concanavalin A in a noncooperative process; however, treatment of the microvilli with the same cytoplasmic effectors does not induce cooperativity. Since previous studies in several systems have suggested an association of nucleotidase with actin-containing microfilaments or the cell cytoskeleton, one explanation for the cooperativity changes is that they result from a change in the association of the enzyme with the cytoskeleton. However, Triton X-100 extractability of nucleotidase is the same for MAT-C1 cells exhibiting cooperative or noncooperative concanavalin A inhibition. Moreover, enzyme from cells exhibiting cooperative inhibition can be extracted into the zwitterionic detergent Zwittergent in a cooperative form, while enzyme exhibiting noncooperative behavior can be extracted into Zwittergent in a noncooperative form. Gel filtration and rate-zonal sucrose density gradient centrifugation showed little discernible size or sedimentation difference between enzyme samples exhibiting noncooperative and cooperative inhibition. These results indicate that changes in the cooperativity of the concanavalin A inhibition of nucleotidase are not a result of changes in the association of the enzyme with the cytoskeleton. These studies emphasize the caution which must be exercised in interpreting the effects of cytoskeletal perturbants on cell surface functions.
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PMID:Transmembrane modulation of the concanavalin A inhibition of 5'-nucleotidase is not due to a direct association of the enzyme with the cytoskeleton. 298 74

5'-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5'-nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E.M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J.P. (1984) Biochem. J. 221, 369-377). Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5'-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5'-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5'-nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver.
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PMID:An improved procedure for purifying 5'-nucleotidase from various sources. Evidence for tissue and species differences in their molecular mass and affinity for F-actin. 299 65

WI-L2 cells (a B-lymphoblastoid cell line) were more resistant than CEM cells (a T-lymphoblastoid cell line) to deoxyadenosine, ara-A (9-beta-D-arabinofuranosyladenine), or ara-C (1-beta-D-arabinofuranosylcytosine) inhibition. This was caused by a difference in the composition of cytosol 5'-nucleotidases between WI-L2 and CEM cells. In intact cells, the endogenous production of deoxyadenosine from WI-L2 cells deficient in adenosine kinase (EC 2.7.1.20) and deoxycytidine kinase (EC 2.7.1.74) was consistently high, despite changes in endogenous adenosine production. Endogenous production of deoxyadenosine from CEM cells deficient in adenosine kinase and deoxycytidine kinase was, however, coordinated with endogenous adenosine production. In broken cells, cytosol dAMPase (2'-deoxyadenosine 5'-monophosphate 5'-nucleotidase) activity of WI-L2 cells was 3-5-fold higher than that of CEM cells. dAMPase activity could be separated from ATP-activated IMPase (inosine 5'-monophosphate 5'-nucleotidase) by gel filtration (molecular weight: dAMPase; 39,000-46,000; ATP-activated IMPase, greater than 150,000). Cytosol ATP-activated IMPase and dAMPase were isolated by phosphocellulose or DEAE-Bio-Gel A chromatography from non-specific phosphatases. The ATP-activated IMPase showed only marginal activity towards dAMP (2'-deoxyadenosine 5'-monophosphate), ara-AMP (9-beta-D-arabinofuranosyladenine 5'-monophosphate), or ara-CMP (cytosine-beta-D-arabinofuranoside 5'-monophosphate), even in the presence of ATP. The activity of ATP-activated IMPase was similar in WI-L2 and CEM cells. dAMPase was separated into two peaks by DEAE-Bio-Gel A chromatography; one of these peaks degraded ara-AMP and ara-CMP. The activities of both peaks from WI-L2 cells were higher than those from CEM cells. These results show that the degradation of dAMP, ara-AMP or ara-CMP was more specific and rapid in WI-L2 than in CEM cells.
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PMID:Differential composition of cytosol 5'-nucleotidases between T and B lymphoblasts. 301 Nov 14

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.
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PMID:The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5'-nucleotidase. 302 68


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