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)

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.
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PMID:Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover. 711 47

Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5'-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg2+-ATPase, (Na+ + K+)-ATPase, gamma-glutamyl transpeptidase, phosphodiesterase I, alkaline phosphatase and xanthine oxidase were also high (12-18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide composition in both light and heavy membranes were similar upon SDS-polyacrylamide gel electorphoresis.
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PMID:Isolation and characterization of plasma membrane from lactating bovine mammary gland. 720 55

The neutrophil plasma membrane has a major role in migration, phagocytosis, and destruction of microorganisms. Neutrophils isolated from blood and mammary secretions were homogenized, and the plasma membrane fraction was isolated on discontinuous sucrose gradient (20, 32, and 50%). Purity of plasma membrane preparation was determined by use of marker enzyme analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the membrane proteins was performed under reducing conditions for polypeptide characterization. The membrane proteins were also labeled with 125I externally, using 1,3,4,6-tetrachloro-3 alpha-6 alpha-diphenylglycouril, and proteins were separated by SDS-PAGE and autoradiographed. Compared with whole cell homogenate, the plasma membrane fraction obtained at the 20/32% interface was enriched for the marker enzymes, 5'-nucleotidase (16-fold), alkaline phosphatase (5.5-fold), and total phosphatase (26-fold). The membrane fraction had minimal specific activity for beta-glucuronidase (0.4-fold), compared with whole cell homogenate. Plasma membrane protein yield was about 500 micrograms/10(9) bovine blood neutrophils. The SDS-PAGE of plasma membrane proteins revealed 25 protein bands, of which 13 were major bands. There were 3 distinct bands (18, 36, and 65 kd) in the plasma membrane-enriched fraction (20/32 interface) that were not seen in other fractions (30/50% and pellet). Further, 125I-labeling identified 5 distinct protein bands (205, 140, 65, 35, and 30 kd). Blood and mammary neutrophils had similar polypeptide patterns, except that 36- and 65-kd bands were more prominent for blood neutrophils than for mammary neutrophils.
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PMID:Isolation and partial polypeptide characterization of bovine neutrophil plasma membranes. 794 18

A glycosylphosphatidylinositol (GPI)-anchored protein, 5'-nucleotidase [EC 3.1.3.5], was released from the membrane of bovine liver by use of phosphatidylinositol-specific phospholipase C (PI-PLC) of Bacillus thuringiensis and purified by several column chromatographies to a homogeneous state. The purified protein has an apparent molecular mass of 61 kDa, as estimated by SDS-polyacrylamide gel electrophoresis. From the partial amino acid sequence of a tryptic peptide, mixed oligonucleotides were synthesized and used to screen a lambda gt11 liver cDNA library, and one positive clone, pE1, was isolated. Since the insert of the clone lacked the NH2-terminal coding region, another lambda gt11 liver cDNA library was screened by using a synthetic probe corresponding to the 5' region of the insert of pE1. Three additional cDNA clones were obtained. Sequencing of these cDNAs revealed an open reading frame that encodes a 574-residue polypeptide with a calculated mass of 63,084 Da. The predicted structure showed two highly hydrophobic stretches at both ends of the protein, like those of rat and human 5'-nucleotidases. The NH2-terminal 26 residues comprise a signal peptide and the COOH-terminal hydrophobic stretch may serve as a signal for the posttranslational GPI modification. An expression vector of the cDNA, pSVNT, was constructed in a mammalian expression vector pSVL and the 5'-nucleotidase activity was transiently expressed in COS-1 cells. The expressed activity was about 8 times higher than the pSVL-transfected control activity. PI-PLC released 45% of the transiently expressed 5'-nucleotidase activity, indicating that the cDNA isolated here encodes this enzyme expressed as a GPI-anchored protein.
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PMID:Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells. 834 Mar 54

Soluble broad spectrum 5'-nucleotidase from human seminal plasma was purified to homogeneity by a combination of (NH4)2SO4 precipitation, affinity chromatography, and gel filtration. The pure enzyme had a specific activity of 4800 nmol min-1 mg-1. SDS-PAGE of purified enzyme preparation revealed a single polypeptide band of 53 kDa and a tetrameric structure of 203 kDa was proposed for the native enzyme. This form had modest preference for AMP as substrate; Mg2+ and Mn2+ were activators of the enzyme although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme, recovered in the nonsedimentable fraction of human seminal plasma, had a pH optimum of 7.5; ATP and ADP were inhibitors of mixed type, Pi was a potent inhibitor at nonphysiological concentrations, and Con A and adenosine 5-[alpha, beta-methylene]diphosphate had no effect on the enzyme activity. The enzyme described here therefore has some unique properties between truly cytoplasmic and membrane-bound derived forms.
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PMID:The isolation from human seminal plasma of a new form of soluble 5'-nucleotidase. 881 36

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5'-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca(2+)-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the mental chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.
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PMID:Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes. 892 Sep 95

Three cytosolic and one plasma membrane-bound 5'-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5'-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5'- and 2'(3')-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.
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PMID:A deoxyribonucleotidase in mitochondria: involvement in regulation of dNTP pools and possible link to genetic disease. 1089 95

The developmental management of 5'-nucleotidase (5nt) expression in Dictyostelium discoideum has provided a focal point for studies of gene regulation at the level of transcription. To identify DNA-protein interactions involved in the 5nt regulation, EMSAs were performed using short oligonucleotides, designed to span a 357bp promoter region. A binding activity (R(f)=0.33) was identified and shown to be specific to the nucleotide sequence between -338 and -309bp relative to 5nt ATG. Characterization of the binding activity, including the effects of salt and temperature, provided insight into the nature and stability of the protein. The protein was purified in a series of chromatographic stages, including DEAE-Sephacel, heparin-Sepharose, DNA affinity, and gel filtration. SDS-PAGE analysis identified a polypeptide with a molecular weight of 70kDa. Mass spectrometry revealed that the purified protein was a putative formyltetrahydrofolate synthase.
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PMID:Identification and purification of a DNA-binding protein interacting with the promoter of 5'-nucleotidase in Dictyostelium discoideum. 1635 68

The expression of proteins in accessory sex gland fluid (AGF) of proven, high use mature Holstein bulls was evaluated. Thirty-seven bulls with documented fertility based on their non-return rates were studied. AGF was obtained by artificial vagina after bulls were surgically equipped with cannulae in the vasa deferentia. Samples of AGF were evaluated by two-dimensional SDS-PAGE, gels stained with Coomassie blue and polypeptide maps analyzed by PDQuest software. A master gel generated by the software representing the best pattern of spots in the AGF polypeptide maps was used as a reference for protein identification. Proteins were identified by Western blots and capillary liquid chromatography-nanoelectrospray ionization tandem-mass spectrometry (CapLC-MS/MS). The product ion spectra were processed using Protein Lynx Global Server 2.1 prior to database search with both PLGS and MASCOT (Matrix Science) software. The entire NCBI database was considered for mass fingerprint matching. An average of 52+/-5 spots was detected in the AGF 2D gels, which corresponded to proteins potentially involved in capacitation (bovine seminal plasma protein-BSP-A1/A2 and A3, BSP 30 kDa, albumin); sperm membrane protection, prevention of oxidative stress, complement-mediated sperm destruction and anti-microbial activity (albumin, clusterin, acidic seminal fluid protein--aSFP, 5'-nucleotidase--5'-NT, phospholipase A2--PLA2); acrosome reaction and sperm-oocyte interaction (PLA2, osteopontin); interaction with the extracellular matrix (tissue inhibitor of metalloproteinase 2, clusterin) and sperm motility (aSFP, spermadhesin Z13, 5'-NT). The 20 spots distinguished in all gels were matched to proteins associated with these functions. Proteins identified by tandem mass spectrometry as ecto-ADP-ribosyltransferase 5 and nucleobindin, never described before in the accessory sex gland secretions, were also detected. In summary, we identified a diverse range of components in the accessory sex gland fluid of a select group of Holstein bulls with documented fertility. Known characteristics of these proteins suggest that they play important roles in sperm physiology after ejaculation.
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PMID:A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls. 1671 41

The phosphodiesterases (PDEs) are a superfamily of enzymes that have multiple roles in extracellular nucleotide metabolism and in the regulation of nucleotide-based intercellular signaling. Here we describe for the first time the isolation and partial characterization of a novel phosphodiesterase from Trimeresurus stejnegeri venom, named TS-PDE, using ion exchange and gel filtration chromatography. The purified TS-PDE is shown to be homogeneous as judged by SDS-PAGE and capillary isoelectric focusing. TS-PDE is a glycoprotein which contains 2.48% carbohydrate. Unlike other PDEs which are usually single polypeptide chain proteins with isoelectric points between 7.5 and 10.5, TS-PDE is a disulfide-linked heterodimer with an isoelectric point of 5.1 and a molecular mass of 100 kDa. The N-terminal amino acids of two chains are valine and serine, respectively. Furthermore, among all identified PDEs, only TS-PDE contains both of endogenous Cu(2+) and Zn(2+) which are essential for its phosphodiesterase activity. The purified TS-PDE exhibits broad phosphodiesterase substrate range with the order of specificity: nicotinamide guanine dinucleotide > ATP > nicotinamide adenine dinucleotide > ADP. The purified TS-PDE shows an exonuclease activity and no contamination with either alkaline phosphatase or 5'-nucleotidase activity. TS-PDE strongly inhibits ADP-induced platelet aggregation in human platelet-rich plasma by hydrolyzing ADP. Altogether, these results indicate that the novel TS-PDE is a unique phosphodiesterase with different structure from the known PDEs.
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PMID:Purification and partial characterization of a novel phosphodiesterase from the venom of Trimeresurus stejnegeri: inhibition of platelet aggregation. 2166 7


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