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)

The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.
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PMID:Properties of 5'-nucleotidase in rat heart sarcolemma. 630 58

The enzymatic activities of the spinal cord membrane phosphatases, (Na,K)ATPase, KpNPPase, 5'-nucleotidase, MgATPase, and MgpNPPase, were determined following contusion injury. Within 30 min after injury, the activities of (Na,K)ATPase, KpNPPase, and 5'-nucleotidase were suppressed at the injury site and remained suppressed for 24 h. Considerable loss of (Na,K)ATPase and KpNPPase activity was seen along the longitudinal axis of the spinal cord for the first 4 h after injury, whereas at 24 h after injury, there was almost complete restitution in the activity of those enzymes. No similar changes in activity were observed for the MgATPase, whereas the activity of the MgpNPPase progressively worsened with time at the injury site. The deduce the pathobiochemical process that accounts for the loss of spinal membrane phosphatase activity, spinal cord membranes were incubated in vitro in the presence of a superoxide anion-generating system, as well as in the presence of trypsin and lysolecithin. It was found that superoxide anions inhibited only the activity of transport phosphatase, KpNPPase, whereas trypsin inhibited the activity of both KpNPPase and MgpNPPase. No inhibition of 5'-nucleotidase was observed by superoxide anions; however, the activity of 5'-nucleotidase was enhanced by trypsin, lysolecithin, or both in concert. These results suggest that the pathobiochemical process that accounts for the loss of spinal membrane phosphatase activity that follows injury can be attributed to the concerted effects of free radical attack as well as the activation of proteolytic and lipolytic enzymes. Inactivation of spinal membrane phosphatase can occur by direct attack of these processes on the membrane or by loss of membrane material, e.g., solubilization.
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PMID:Effect of traumatic injury on membrane phosphatase activity in cat spinal cord. 631 25

Protein synthesis in isolated yeast mitochondria incubated in the presence of GTP is stimulated 2-fold by addition of dialyzed postpolysomal supernatant (S-150) at the start of the incubation. Incubation of the yeast S-150 with 5'-nucleotidase had no effect on the stimulatory activity suggesting that the increased protein synthesis does not result from guanine nucleotides. A partial purification of the protein factors which stimulate mitochondrial protein synthesis has been accomplished by chromatography on Sephacryl S-200. Stimulatory activity was eluted in two peaks, one in the 40,000 to 80,000 molecular weight range and a broad peak with a molecular weight of less than 10,000. Stimulation of mitochondrial protein synthesis by the low molecular weight activator fraction was proportional to the concentration of protein added and abolished by trypsin treatment suggesting that the low molecular weight activator is a protein(s). The rate of mitochondrial protein synthesis in the presence of activator, was linear for 40 min, while that in the presence of GTP was linear for only 20 min, suggesting that the activator and GTP stimulate protein synthesis by different mechanisms. Analysis of the products of the stimulated mitochondrial protein synthesis by gel electrophoresis revealed that the activator increased equally the labeling of all products. These results indicate that low molecular weight proteins present in the cytosol regulate mitochondrial protein synthesis.
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PMID:Partial purification of cytosolic proteins which control yeast mitochondrial protein synthesis. 702 43

An aqueous extract of normal human skin has been shown to contain an inhibitor of certain cell mediated immune reactions. In this report, the effect of the inhibitor on cell membrane markers and antibody dependent cellular cytotoxicity was determined. Significant diminution of E rosette formation was demonstrated using as little as 0.6 microgram of the skin fraction (p less than .02). Fc receptors for both IgG and IgM were reduced by 46-96% of controls in the presence of the skin inhibitor. On the other hand, no effect on the detection of the complement receptor or surface immunoglobulin was observed, indicating some specificity of binding. In addition, the antibody dependent cell-mediated cytotoxic reaction was inhibited on the skin extract. It was shown that the inhibitor interacted with the lymphocytes, not the antibody or target cells. No effect was detectable when the skin fraction was added after the interactions of effector cells, antibody, and target cells had occurred. This was in contrast to PHA-induced cytotoxicity which could be inhibited following the preincubation of the lymphocytes with the mitogen. Thus there appears to be 2 mechanisms by which the skin fraction interferes with cellular responses: inhibition of antibody binding to Fc receptors, and interference with a step in cellular activation following mitogen stimulation. Analysis of the extract showed the inhibitor was inactivated by trypsin, and did not contain sialic acid, 5'-nucleotidase of beta-N-acetylglucosaminidase, and thus was not associated with membrane or lysosomal enzymes.
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PMID:Inhibition of cell-mediated immune reactions by an aqueous extract of normal human skin. 710 62

Viable and homogeneous endothelial cells were obtained from isolated guinea pig hearts by application of a special perfusion technique of the coronary system with an isotonic collagenase-trypsin solution and subsequent purification of the dissociated cells by Percoll density gradient centrifugation. The coronary endothelial cells were grown in tissue culture for periods up to 7 months. Serial passage proved to be possible. During logarithmic growth, generation time was found to be 18 h; it could be reduced to 16 h by addition of thrombin to the culture medium. Light, phase contrast and scanning electron microscopy as well as autoradiography revealed that cultured coronary endothelial cells grew as strict monolayers of closely apposed, polygonal large cells. By scanning electron microscopy, it could be demonstrated that the morphology of the cultured cells changes characteristically during attachment of the cells to their substratum. The changes observed were very similar to those of proliferating endothelial cells of isolated coronary vessels kept in organ culture. According to transmission electron microscopy studies, cultured coronary endothelial cells proved to contain only an extremely small number of Weibel-Palade bodies. Nucleoside phosphorylase (EC 2.4.2.5.) and 5'-nucleotidase (EC 3.1.3.5.) were identified in freshly isolated as well as in cultured endothelial cells. Their specific and total activities proved to be much higher than in myocardial tissue, thus indicating a prominent role of nucleotide metabolism in the coronary endothelium.
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PMID:Isolation, identification, and continuous culture of coronary endothelial cells from guinea pig hearts. 728 45

Hepatic 5'-nucleotidases of vertebrates were investigated for localization in the lysosomes and the plasma membrane, microheterogeneity of the glycosylphosphatidylinositol (GPI)-anchor moiety and minimal requirement of the C-terminal signal peptide for GPI attachment. Using PIPLC of Bacillus thuringiensis and subcellular fractionation by Percoll gradient centrifugation, we found that chicken liver 5'-nucleotidase can be transferred from plasma membrane to lysosomes in the GPI-anchored or soluble form. Bovine liver ecto 5'-nucleotidase was solubilized by PIPLC, purified to a homogeneous state, and analyzed for the structures of GPI-anchor isoforms by HPLC and ESI-MS in combination with glycosidase treatments, after peptide-bond cleavage by CNBr or trypsin. Several isomers of the GPI anchor were thus characterized; major components contained two phosphorylethanolamine residues, whereas the component containing three phosphorylethanolamine residues was present only as a small percentage of the total. The cleavage/attachment site of the GPI anchor in the C-terminal of 5'-nucleotidase was shown to be Ser523. The peptide region cleaved off at the posttranslational processing has a length of 25 amino acid residues which contains a hydrophobic stretch of 17 amino acids. By site-directed mutagenesis, we determined the minimal length of the hydrophobic peptide to be 13 amino acids for expression of 5'-nucleotidase as a GPI-anchored form on the COS cell surface. When peptide length was shortened to less than 13 amino acids, the expressed enzyme was not sorted to the cell surface but present within, or secreted out of the cells.
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PMID:Studies on the GPI-anchored enzyme, hepatic 5'-nucleotidase. Microheterogeneity of the anchor, processing and localization. 808 Dec 55

In our study, 5'-nucleotidase was released from bovine liver by the treatment with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and purified to a homogeneous state by concanavalin A-Sepharose and (diethylaminoethyl)-Toyopearl column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified 5'-nucleotidase were then cleaved by cyanogen bromide (CNBr), and then inositol phosphoglycan-containing C-terminal peptides (IPG peptides) were separated by C18 reverse-phase liquid chromatography and analyzed by peptide sequencer, amino acid analyzer, gas chromatography (GC), and GC-mass spectrometry (MS). Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA [Suzuki et al. (1993) J. Biochem. (Tokyo) 113, 607-613] is revealed to be the C-terminal amino acid to which a glycosylphosphatidylinositol is anchored. Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS. Eight different molecular weight (MW) species of CNBr-cleaved IPG peptides were detected. Three fractions of CNBr-cleaved IPG peptides were separately treated by trypsin, and trypsinized IPG peptides were purified by C18 reverse-phase liquid chromatography. Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8, and 1994.5) were detected by ESI-MS. Together with sequential exoglycosidase treatment and quantitative analysis of sugar moieties by GC and GC-MS, microheterogeneity in the structures of these five glycosylphosphatidylinositol (GPI) anchor species was determined. The common core structure was ethanolamine phosphate-mannose-mannose-mannose(-ethanolamine phosphate)-glucosamine-myoinositol phosphate. Variations observed in additional mannose, N-acetylhexosamine, and ethanolamine phosphate moieties form this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Microheterogeneity in glycosylphosphatidylinositol anchor structures of bovine liver 5'-nucleotidase. 830 28

The development of satisfactory cell culture models for the study of parathyroid hormone (PTH)-induced inhibition of Pi transport has proven difficult. Using subcellular fractionation techniques we investigated the response of primary cultures of rat proximal tubular cells to PTH-(1-34). Specific binding of 125I-bPTH-(1-34) occurred at 2 degrees C. After 5 min of rewarming, trypsin-releasable radioactivity decreased from 90 to 50%, indicating internalization of the ligand. Cell disruption, followed by density centrifugation with 17% Percoll either directly after binding at 2 degrees C or post-rewarming for 20 min, showed a shift of 125I label from the plasma membrane (5'-nucleotidase) to lysosomal fractions (beta-D-glucosaminidase), confirming the sequential occurrence of cell surface binding, internalization and transport to lysosomes of 125I-bPTH-(1-34). Reculture at 37 degrees C revealed steady accumulation of trichloroacetic acid-soluble radioactivity in the medium, indicating degradation of 125I-bPTH-(1-34). Phosphate transport in the absence of sodium was minimal. Incubation of the cells with bPTH-(1-34) resulted in up to 50% inhibition of sodium-dependent phosphate transport. Prior phosphate depletion abrogated the response to PTH.
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PMID:Parathyroid hormone transport effects and hormonal processing in primary cultured rat proximal tubular cells. 834 17

We show that Escherichia coli produce a factor that inhibits the activity of tyrosine and serine/threonine protein kinases. The factor is a protein found in the periplasmic compartment and is also secreted into the culture medium. Using a particle concentration fluorescence immunoassay specific for tyrosine kinase activity and inhibition of the tyrosine kinase p56(lck), we purified this factor to apparent homogeneity. Analysis of trypsin-digested fragments by mass spectrometry identified the inhibitor as the bacterial periplasmic protein UDP-sugar hydrolase, an enzyme with potent and nonspecific 5'-nucleotidase activity. Overexpression of the enzyme in bacteria leads to coordinate increases in both 5'-nucleotidase and p56(lck) inhibitory activity, confirming the identity of the inhibitor. The kinase inhibitory activity appears to be due to the formation of adenosine, which we show is inhibitory for p56(lck), cAMP-dependent protein kinase, and casein kinase. Overexpression of UDP-sugar hydrolase leads to an increase in the recovery of enteropathogenic E. coli following infection of HeLa cell monolayers and corresponding alterations in tyrosine-phosphorylated host proteins. These results suggest that UDP-sugar hydrolase may be an important factor affecting host cell function following intracellular bacterial infection.
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PMID:Identification of a bacterial inhibitor of protein kinases. Mechanism and role in host cell invasion. 879 49

In the present study, some biochemical properties and pathological effects of Daboia russelli venom from Burdwan district of West Bengal, eastern India are presented. The clinical features of Russell's viper envenomation observed in patients admitted to Burdwan Medical College & Hospital are also reported. In vitro, whole venom exerts strong trypsin inhibitory, phospholipase A2 and procoagulant activities in addition to moderate adenosine monophosphatase and adenosine triphosphatase activities. Lethality (LD50) of this venom sample is 0.7 mg kg (i.v.) of mice. Significant local tissue damaging effects including edema, hemorrhage and necrosis are observed in experimental animal models. An increase in the level of serum enzymes, such as aspartate transaminase, alkaline phosphatase, creatine phosphokinase, lactate dehydrogenase after D. russelli venom injection in albino rats is indicative of cell or tissue damage. High incidence of intravascular hemolysis in addition to hemostasis, haemoptysis and haematuria are observed as the most prominent features of RVV envenomation from this part of India. The present study reinforces the hypothesis that variation in the venom composition of RVV from eastern India with respect to venom samples of Russell's vipers from other parts of India is responsible for the differences in the clinical manifestation in patients from eastern India.
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PMID:Some biochemical properties of Russell's viper (Daboia russelli) venom from Eastern India: correlation with clinico-pathological manifestation in Russell's viper bite. 1066 98


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