Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An assay for thymidine substitution by iododeoxyuridine (IdUrd) using reversed-phase high-performance liquid chromatography (HPLC) has been developed. Three principal steps in this procedure are: extraction of DNA from cell or tissues, hydrolysis of DNA into deoxynucleosides and separation using HPLC. Approximately 1 microgram of DNA was recovered from 10(5) cells by phenol extraction, and subjected to hydrolysis into deoxynucleosides which required a three-stage DNA digestion using enzymes DNAse I. phosphodiesterase I and alkaline phosphatase. The deoxynucleosides were separated on the Microsorb C18 column with isocratic elution; 90-100% of the DNA was recovered as deoxynucleosides on the column. The method was used to determine quantitatively the percent IdUrd substitution of thymidine in Chinese hamster lung cells in vitro and BA1112 rhabdomyosarcoma in WAG/Rij rats perfused with IdUrd. It was possible to determine the thymidine substitution by IdUrd as small as 1% using a few micrograms of DNA. The close correspondence between the percent substitutions determined by HPLC and those determined by radioactive assay using [125I]-labelled IdUrd, confirmed the accuracy of our HPLC method. The HPLC analysis is especially suitable for the determination of percent IdUrd substitution of thymidine in tissue biopsies from animals used in in vivo experiments or humans undergoing radiation treatment.
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PMID:A method for determination of iododeoxyuridine substitution of thymidine using reversed-phase high-performance liquid chromatography. 200 16

Conditions favouring protein phosphorylation and dephosphorylation are examined for their effects on activity and charge heterogeneity of the rat gastric mucosal histidine decarboxylase. Incubation of gastric supernatant with various combinations of ATP, Mg2+, cyclic AMP and protein kinase under the blockade of endogenous phosphodiesterase and phosphatase fails to alter significantly enzyme activity as assayed with or without pyridoxal 5'-phosphate. Similar results are found with the purified enzyme. No change occurs in the distribution of activity between the charged forms. In contrast, treatment with alkaline phosphatase both inactivates the enzyme with preservation of heterogeneity, full reactivation being achieved by pyridoxal 5'-phosphate, and reduces the number of forms and converts forms II and III to form I with preservation of the catalytic potentialities. The data suggest that the enzyme heterogeneity may be related in part to the phosphorylation state; the possibility that the gastric enzyme is susceptible to several post-translational modifications is discussed.
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PMID:Relationship between the multiple forms of rat gastric histidine decarboxylase: effects of conditions favouring phosphorylation and dephosphorylation. 215 9

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 31 samples of venom from three species of Agkistrodon (A. bilineatus, A. contortrix and A. piscivorus) and 10 venom samples from five other related species belonging to the same tribe of Agkistrodontini were examined. 2. The results indicate that interspecific differences in certain biological activities of the Agkistrodon venoms are more marked than individual variations of the activities, and that these differences can be used for differentiation of the species. Particularly useful for this purpose are the phosphodiesterase, arginine ester hydrolase and anticoagulant activities of the venoms. 3. Venoms of the subspecies of A. contortrix and A. piscivorus do not differ significantly in their biological activities.
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PMID:A comparative study of the biological activities of venoms from snakes of the genus Agkistrodon (moccasins and copperheads). 215 74

A new method for the determination of the level of DNA methylation was established. The method involves enzymatic hydrolysis of DNA by nuclease P1 and bacterial alkaline phosphatase, and separation of the resulting deoxyribonucleosides by HPLC. By this method, DNA was hydrolysed completely to the five deoxyribonucleosides and the complete base composition was determined. Pairing bases were shown to occur in similar amounts, and analysis could be performed on as little as 1 microgram of DNA with a high degree of reproducibility. Among other enzymes hitherto used in order to hydrolyze DNA, micrococcal nuclease, phosphodiesterase II and nuclease P1 have been shown to cause deamination of deoxyadenosine, while deoxyribonuclease I, phosphodiesterase I and bacterial alkaline phosphatase have been shown to be sensitive to contamination by RNA, and to release 5-methyldeoxycytidine at a slower rate than the other four deoxyribonucleosides. Neither of these effects was seen with the new method.
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PMID:Employment of hydrolytic enzymes in the study of the level of DNA methylation. 216 80

Alkaline phosphatase and 5'-nucleotidase are covalently linked to phosphatidylinositol in bovine fat globule membrane, as demonstrated by their release following treatment with phospholipase C specific for phosphatidylinositol. The failure of this treatment to liberate phosphodiesterase I may indicate that it has a variant linkage resistant to release. In a test of exposure at the membrane surface, alkaline phosphatase and phosphodiesterase I, but not 5'-nucleotidase, were released from fat globule membrane by treatment with proteinase K. These apparent differences in accessibilities of membrane surface proteins suggest that attachment to phosphatidylinositol does not necessarily impart greater exposure to proteins with which it is linked.
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PMID:Differential release of proteins from bovine fat globule membrane. 216 62

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

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 26 samples of venoms of 13 taxa of Vipera were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate the presence of certain common characteristics among the venoms, particularly if V. russelli is excluded from the comparison. The results also support the recently proposed reassignment of V. russelli to a separate genus. 3. The data show that information on venom biological properties can be used for differentiation of venoms of many species of Vipera. Particularly useful for this purpose are the protease, phosphodiesterase, phospholipase A and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.
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PMID:A comparative study of the biological properties of venoms from snakes of the genus Vipera (true adders) 217 67

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


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