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:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Liver plasma membranes originating from the sinusoidal, lateral and canalicular surface domains of hepatocytes were covalently labelled with sulpho-N-hydroxysuccinamide-biotin. After solubilization in Triton X-114, treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), two-phase partitioning and 125I-streptavidin labelling of the proteins resolved by PAGE, six major polypeptides (molecular masses 110, 85, 70, 55, 38 and 35 kDa) were shown to be anchored in bile canalicular membrane vesicles by a glycosyl-phosphatidylinositol (G-PI) 'tail'. 2. Permeabilized 'early' and 'late' endocytic vesicles isolated from liver were also examined. Two polypeptides (110 and 35 kDa) were shown to be anchored by a G-PI tail in 'late' endocytic vesicles. 3. Analysis of marker enzymes in bile-canalicular vesicles treated with PI-PLC showed that 5'-nucleotidase and alkaline phosphatase, but not leucine aminopeptidase and ecto-Ca2(+)-ATPase activities were released from the membrane. A low release and recovery of alkaline phosphodiesterase activity was noted. The cleavage from the membrane of 5'-nucleotidase as a 70 kDa polypeptide was confirmed by Western blotting using an antibody to this enzyme. 4. Antibodies raised to proteins released from bile-canalicular vesicles by PI-PLC treatment, and purified by partitioning in aqueous and Triton X-114 phases, localized to the bile canaliculi in thin liver sections. Antibodies to proteins not hydrolysed by this treatment stained by immunofluorescence the sinusoidal and canalicular surface regions of hepatocytes. 5. Antibodies generated to proteins cleaved by PI-PLC treatment of canalicular vesicles were shown to identify, by Western blotting, a major 110 kDa polypeptide in these vesicles. Two polypeptides (55 and 38 kDa) were detected in MDCK and HepG-2 cultured cells. 6. Since two of the six G-PI-anchored proteins targeted to the bile-canalicular plasma membrane were also detected in 'late' endocytic vesicles, the results suggest that a junction where exocytic and endocytic traffic routes meet occurs in a 'late' endocytic compartment.
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PMID:Priority targeting of glycosyl-phosphatidylinositol-anchored proteins to the bile-canalicular (apical) plasma membrane of hepatocytes. Involvement of 'late' endosomes. 217 97

We have conducted studies to obtain practical knowledge regarding the stability, digestion, and analytical determination of the content of 8-hydroxy-2-deoxy-guanosine (8-OHdG) in oxidatively damaged DNA. Utilizing H2O2 plus uv light to form oxidatively damaged DNA, we found that storage of the DNA at -20 degrees C at alkaline pH caused a significant loss of 8-OHdG, whereas storage at -20 degrees C at neutral or acidic pH prevented loss of 8-OHdG. The 8-OHdG within DNA is stable at 100 degrees C for at least 15 min. Formation of 8-OHdG within DNA using uv light and H2O2 as a hydroxyl free radical-generating system yields the highest amounts when low levels of phosphate buffer are used; but the use of Tris or citrate buffers causes a lower yield of 8-OHdG because these buffers act as scavengers for the hydroxyl free radicals. Independent assessment of hydroxyl free radical flux by the use of salicylate trapping allows assessment of competitive radical reactions. Ethanol washing of plastic microfuge tubes prior to DNA enzymatic digestion improved the yield of 8-OHdG and reduced the variability between samples. Digestion of the oxidatively damaged DNA by the use of a method involving DNase I, endonuclease, phosphodiesterase, and alkaline phosphatase produced the highest yield of 8-OHdG.
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PMID:Conditions influencing yield and analysis of 8-hydroxy-2'-deoxyguanosine in oxidatively damaged DNA. 222 56

3'-Azido-2',3'-dideoxyuridine (AzdU, CS-87) is a potent inhibitor of human immunodeficiency virus replication in human peripheral blood mononuclear cells (PBMC) with limited toxicity for human bone marrow cells (BMC). In the present study, metabolism of AzdU was investigated in human PBMC and BMC after exposure of cells to 2 or 10 microM [3H]AzdU. 3'-Azido-2',3'-dideoxyuridine-5'-monophosphate (AzdU-MP) was the predominant metabolite, representing approximately 55 to 65% of intracellular radioactivity in both PBMC and BMC at all times. The AzdU-5'-diphosphate and -5'-triphosphate intracellular levels were 10- to 100-fold lower than the AzdU-MP levels and, of note, AzdU-5'-triphosphate was not detected in human BMC. Using anion exchange chromatography, a new peak of radioactivity, distinct from any known anabolites, was detected. This chromatographic peak was found to be resistant to alkaline phosphatase but was hydrolyzed by 5'-phosphodiesterase, yielding AzdU-MP. Incubation of [3H]AzdU and D-[1-14C]glucose in PBMC and BMC produced a double-labeled peak with the same retention time as the anabolite, suggesting formation of a hexose derivative of AzdU. A novel high performance liquid chromatography method was developed that allowed for the separation of nucleosides, nucleotides, and carbohydrate derivatives thereof. Using this highly specific method, the putative AzdU-hexose actually was separated into two chromatographic peaks. These novel metabolites were identified as 3'-azido-2',3'-dideoxyuridine-5'-O-diphosphoglucose and 3'-azido-2',3'-dideoxyuridine-5'-O-diphospho-N-acetylglucosamine. Following 48 hr of incubation with [3H] AzdU, as much as 20 and 30% of these AzdU metabolites accumulated in PBMC and BMC, respectively. When AzdU was removed from the cell cultures, intracellular AzdU diphosphohexose concentrations decayed in a monophasic manner, with an elimination half-life of 14.3 hr. By 48 hr, levels of 0.3 pmol/10(6) cells were still detected, reflecting a gradual anabolism of these metabolites. Elimination of AzdU-MP and AzdU-5'-diphosphate was characterized by a two-phase process, with a short initial half-life of 0.83 and 0.24 hr and a long terminal half-life of 14.10 and 8.24 hr, respectively. Similar diphosphohexoses of deoxyuridine (dUrd) were also detected in human PBMC and BMC after exposure to [3H]dUrd, suggesting that dUrd derivatives are metabolized in a similar manner. In summary, the discovery of novel metabolic pathways for dUrd analogs demonstrates that AzdU has unique metabolic features that may contribute to the low toxicity of this anti-HIV agent in human BMC and also affect its mechanism of action.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cellular metabolism of 3'-azido-2',3'-dideoxyuridine with formation of 5'-O-diphosphohexose derivatives by previously unrecognized metabolic pathways for 2'-deoxyuridine analogs. 225 Jun 66

Complete replacement of the nucleotide on the exchangeable binding site of purified calf brain tubulin by the non-hydrolyzable GTP-analogue guanylyl-(beta,gamma-methylene)diphosphonate (GMPPCP) has been achieved by treatment of tubulin-GDP with phosphodiesterase-free alkaline phosphatase. GMPPCP binds to tubulin with a low affinity relative to GTP or GDP. Binding of the analogue is linked to magnesium ion concentration and, like the binding of other guanine nucleotides, is promoted by high concentrations of glycerol. The complex of pure tubulin and GMPPCP readily assembles at 37 degrees C into microtubules or curled ribbons of protofilaments, depending on buffer composition. Assemblies are cold-reversible at 0-2 degrees C, and multiple reversible assemblies can be observed during repeated heating/cooling cycles.
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PMID:Interactions of tubulin with guanylyl-(beta-gamma-methylene)diphosphonate. Formation and assembly of a stoichiometric complex. 233 45

Polyclonal antibodies to native alkaline phosphatase and to native 5'-nucleotide phosphodiesterase were found to strongly cross-react with both enzymes. The antibodies also cross-react with both denatured enzymes, with glycopeptides from 5'-nucleotide phosphodiesterase, and with the oligosaccharides remaining after Pronase E digestion of the phosphodiesterase. They do not cross-react with either enzyme after their oligosaccharides have been modified or removed by periodate or trifluoromethanesulfonic acid treatment. Antibodies to denatured 5'-nucleotide phosphodiesterase do not bind to the native phosphodiesterase or alkaline phosphatase but do cross-react with denatured alkaline phosphatase even after removal or modification of the carbohydrate moieties. These results suggest that antibodies to denatured 5'-nucleotide phosphodiesterase may recognize amino acid sequence homology between alkaline phosphatase and 5'-nucleotide phosphodiesterase. However, antibodies to native enzymes apparently recognize cross-reactive determinants of the native enzymes which are carbohydrate in nature. This is the first report of antimammalian alkaline phosphatase antibodies which recognize the carbohydrate moieties of the enzyme.
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PMID:Alkaline phosphatase and 5'-nucleotide phosphodiesterase from bovine intestine are cross-reactive. 241 45

cAMP has been shown to be a second messenger in the release of many hormones and other secretory products. To determine whether cAMP also plays a role in the mechanism of release of human placental lactogen (hPL), we examined the effects of (Bu)2cAMP, isobutyl methylxanthine, forskolin, and cholera toxin on the acute release of hPL from an enriched fraction of hPL-producing trophoblast cells. Static cultures of trophoblast cells exposed to (Bu)2cAMP (5 mM) for 2 h released 2.6 times as much hPL as control cells (P less than 0.01) during the first 0.5 h of exposure. The increase in hPL release was followed by a decrease rate of release during the subsequent 1.5 h. Perifused trophoblast cells (1.5 X 10(6) exposed to 5 mM cAMP for 20 min released 3.2 times as much hPL as control cells. The rate of hPL increased markedly during the first 10 min of exposure, rapidly decreased toward control values during the remainder of the exposure period, and then declined to a subnormal rate for the next 30 min before returning to normal to control values. (Bu)2cAMP, however, had no acute effects on the release of human CG or the release of the cytosolic enzymes alkaline phosphatase and lactic dehydrogenase. The phosphodiesterase inhibitors theophylline (5 mM) and isobutyl methylxanthine (0.5 mM) and the adenylate cyclase activators forskolin (5 micrograms/ml) and cholera toxin (25 micrograms/ml) stimulated hPL release by 75-95%. These results strongly suggest that cAMP is a second messenger in the acute release of hPL.
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PMID:Cyclic adenosine-3',5'-monophosphate stimulates the acute release of placental lactogen from human trophoblast cells. 243 14

The chemical nature of association of RNA in immunoprecipitates of human SS-B/La ribonucleoprotein, an autoantigen expressed in various autoimmune disorders, was investigated. A fraction of RNA associated with SS-B/La immunoprecipitates was readily dissociated by SDS-polyacrylamide gel electrophoresis, yielding four main subfractions, R1-4, with chain lengths in the range of 90-130 nucleotides (R4), 140-175 nucleotides (R2 and R3) and above 200 nucleotides (R1). Moreover, the immunoreactive protein component, migrating with a molecular mass of 49 kDa, contained a very tightly bound RNA co-migrating with the protein unless the protein was proteolytically degraded. Most of the RNA molecules in this fraction, represented by about 20 components, had a free 3'-terminus but a blocked 5'-terminus and showed chain lengths between 10 and 125 nucleotides. After pretreatment with alkaline phosphatase and a mixture of ribonucleases T1 + T2 + A, adenosine 3',5'-biphosphate (pAp) was liberated by phosphodiesterase (Crotalus durissus) as the blocked 5'-end of the RNA. The chemical nature of the blockage was revealed after alternative treatment of the protein-pAp component with phosphodiesterase or nuclease S7 followed by acid hydrolysis and phosphoamino acid analysis which showed that a threonine residue must be directly involved in the RNA-protein linkage of 49 kDa SS/La antigen, indicating the presence of a covalent threonine-pAp bond.
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PMID:Human SS-B/LA autoantigen contains a covalent protein-RNA linkage. 244 50

The effect of synthetic human parathyroid hormone (hPTH) on the formation of matrix vesicles (MV), and on the rate of cell division, production of cellular alkaline phosphatase (AP) and protein by primary cultures of chicken epiphyseal growth plate hypertrophic chondrocytes was investigated. Addition to serum-containing or serum-free media of physiological levels of hPTH, in a range from 0.1 to 10 nM, caused a progressive decrease in the formation of AP-rich MV. However, studies on incorporation of [3H]choline into MV indicate that MV formation per se was not significantly decreased. hPTH was found to markedly decrease the expression of cellular AP, accompanied by an increase in cell division [( 3H]thymidine incorporation) and protein synthesis. Since these effects of hPTH were augmented by 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, and mimicked by the cAMP analogue N6,O2'-dibutyryl-adenosine 3',5'-cyclic-monophosphate (DBcAMP), the findings clearly indicate that hPTH was acting through the classic cAMP-mediated mechanism. Inasmuch as elevation of AP in growth plate chondrocytes coincides with MV formation, maturation and hypertrophy of the cells, and induction of mineralization, the stimulation of cell division and suppression of cellular AP indicates that hPTH would cause the cells to revert to a less differentiated state. Thus, elevation in PTH, which results from lowered circulating levels of Ca2+, should inhibit mineral deposition in the growth plate. This may be a physiological protective mechanism to prevent a further drain on serum Ca2+.
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PMID:Effect of synthetic human parathyroid hormone on the levels of alkaline phosphatase activity and formation of alkaline phosphatase-rich matrix vesicles by primary cultures of chicken epiphyseal growth plate chondrocytes. 246 54

The effects of prostaglandins (PGs) on the induction of alkaline phosphatase (ALP) were investigated in osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. Prostaglandin E2 (PGE2) stimulated ALP activity in the cells in a dose-dependent fashion with a maximal effect which was about twice that in the control cells at concentrations of 100-500 ng/ml. Actinomycin D and cycloheximide inhibited the stimulative effect of PGE2 on ALP activity in the cells. PGE2-induced and native ALPs in the cells were of the same type as that in adult mouse calvaria, being heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive. Isobutyl methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, stimulated the inductive effect of PGE2 on ALP activity at 0.1 mM, at which concentration IBMX alone had little effect on the activity. PGE2 also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 100 ng/ml. PGE1, PGF1 alpha, and PGF2 alpha (primary PGs like PGE2) increased the activity. Our present results suggest that PGs stimulate the differentiation of osteoblasts and are involved in bone formation in vivo, as well as in bone resorption.
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PMID:Inductive effects of prostaglandins on alkaline phosphatase in osteoblastic cells, clone MC3T3-E1. 258 42

During early sexual development in Dictyostelium discoideum cell and pronuclear fusion are negatively regulated by an endogenous autoinhibitor. Here, the autoinhibitor was partially purified from the culture medium and found to inhibit both cell and pronuclear fusion while augmenting gamete numbers. These developmental effects suggested that calmodulin might be an intracellular target for the autoinhibitor. In support of this data, the partially purified autoinhibitor inhibited the calmodulin-dependent activation of phosphodiesterase in a dose-dependent manner, but had no effect on either a calmodulin-insensitive form of phosphodiesterase or the calmodulin-independent enzymes acid and alkaline phosphatase. Thus, the autoinhibitor of sexual development in Dictyostelium discoideum appears to regulate cell and pronuclear fusion at least in part by a direct effect on calmodulin.
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PMID:The autoinhibitor of cell fusion in Dictyostelium inhibits calmodulin. 259 Jan 96


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