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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Small oligonucleotides from DNA and RNA have been separated according to their base composition by high-performance anion-exchange liquid chromatography on Partisil-10 SAX using triethylammonium acetate buffer as the eluent. Fifteen of the 16 possible deoxydinucleoside monophosphates and all 16 dinucleoside monophosphates have been separated. All pairs of sequence isomers were all resolved. The 15 commercially available deoxydinucleotides were resolved into 13 fractions. A good resolution of deoxytrinucleoside diphosphates isolated from an alkaline phosphatase-Mg2+-activated DNase I digest of calf thymus DNA was achieved by this technique. A large number of sequence isomers could be fully separated. The base sequence of the eluted individual constituents has been determined by their hydrolysis with snake venom and spleen phosphodiesterase followed by high-performance liquid chromatographic analysis of the nucleotides released. The eight trinucleoside diphosphates isolated from an alkaline phosphatase-pancreatic RNase digest of yeast RNA have also been separated according to base composition. Their sequence was determined as above. The described technique is fast and gave very good separation. Most of the sequence isomers could be separated. Moreover, the eluent triethylammonium acetate can easily be removed from column effluents by freeze-drying in order to facilitate subsequent sequence analysis of the eluted compounds. The observed elution orders of the sequence isomers obey certain rules which are discussed in detail.
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PMID:Separation of small DNA and RNA oligonucleotides by high-performance anion-exchange liquid chromatography. 9 13

Aqueous solutions of DNA were gamma-irradiated in the presence and absence of oxygen and enzymatically hydrolysed by the combined action of pancreatic deoxyribonuclease (DNase I), snake-venom phosphodiesterase (PDE I), spleen phosphodiesterase (PDE II) and alkaline phosphatase. In contrast to unirradiated DNA, which is fully hydrolysed to nucleosides by these enzymes, gamma-irradiated DNA yields a series of oligonucleotides. Their isolation might enalbe the future identification of the chemical nature of DNA lesions.
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PMID:Enzymatic digestion of DNA gamma-irradiated in aqueous solution separation of the digests by ion-exchange chromatography. 21 Jan 33

The DNase I sensitivity of three different chromatin regions in mouse testicular cells was analysed by in situ nick translation with biotin-dUTP combined with various counterstaining techniques. The regions were: (i) the constitutive centromeric heterochromatin, (ii) an interstitial C-band positive insertion on chromosome 1, Is(HSR1;C5)1Lub, and (iii) the chromatin containing rDNA (designated nucleolar chromatin herein). Incorporated biotin was detected either by the horseradish peroxidase reaction with diaminobenzidine (DAB) or the alkaline phosphatase reaction with fast red. The latter resulted in a water insoluble red precipitate, which was easily removable by any organic solution thus allowing the application of various counterstaining protocols. DNase I sensitivity of the three chromatin regions was screened in different cell types of the mouse testis. The interstitial Is(HSR) region was highly DNase I sensitive when it was recognizable by strong mithramycin fluorescence. The centromeric heterochromatin was DNase I resistant when it was compacted into microscopically visible chromosomal structures (mitosis, pachytene, metaphase I and II). In interphase nuclei from Sertoli cells and spermatogonia it became highly DNase I sensitive. In round spermatids it displayed medium DNase I sensitivity. Nucleolar chromatin was not labelled by in situ nick translation when silver staining demonstrated strong protein production. Sperm cells were highly DNase I sensitive from stages 11 to 15, but resistant as mature spermatozoa.
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PMID:Nonradioactive in situ nick translation combined with counterstaining: characterization of C-band and silver positive regions in mouse testicular cells. 169 89

A 32-P-postlabeling assay has been developed that permits detection of several radiogenic base and sugar lesions of DNA at the femtomole level. The technique is based on the inability of DNase I and snake venom phosphodiesterase to cleave the internucleotide phosphodiester bond immediately 5' to the site of damage so that complete digestion of irradiated DNA with these nucleases and alkaline phosphatase yields lesion-bearing "dinucleoside" monophosphates. Because these fragments contain an unmodified nucleoside at the 5'-end of each molecule, they can be readily phosphorylated by T4 polynucleotide kinase and [gamma-32P]ATP and analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC. We observed a linear induction of total damage in DNA irradiated with 5-50 Gy. Virtually no damage was detected when the DNA was irradiated in solution containing 1 M DMSO, implicating hydroxyl radicals in the formation of these lesions. Evidence for the presence of thymine glycols and phosphoglycolate groups came from (i) a comparison of the radiation-induced products with those produced by OsO4 and KMnO4 and (ii) incubation of irradiated DNA with Escherichia coli endonuclease III and exonuclease III before analysis by the postlabeling procedure. This was confirmed by comigration of the radiogenic products with chemically synthesized markers. G values of 0.0022 and 0.0105 mumol J-1 were obtained for thymine glycol and phosphoglycolate production, respectively. The identity of the 5'-nucleotide of each isolated compound was obtained by nuclease P1 digestion. This analysis of nearest-neighbor bases to thymine glycols and phosphoglycolates indicated a nonrandom interaction between radiation-induced hydroxyl radicals and DNA.
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PMID:32P-postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini. 184 59

Oxidative modification of genetic material has been implicated as a factor in carcinogenesis, particularly during promotion and progression, and therefore there is a need for sensitive detection of oxidized DNA bases. We developed a method that can be applied to DNA isolated from any source and used to simultaneously quantify oxidized nucleosides without a need to prelabel the DNA or use destructive hydrolytic procedures. This method is based on: (a) enzymatic DNA digestion; (b) HPLC separation of the resultant nucleosides; (c) acetylation of the oxidized nucleosides with [3H]Ac2O (acetic anhydride); (d) removal of the radioactive debris; and (e) quantitative analysis of tritiated nucleoside acetates by HPLC. Enzymatic DNA digestion was optimized using DNase I in the presence of Mg2+ (pH 7), followed by nuclease P1 in the presence of Zn2+ (pH 5.1) and alkaline phosphatase (pH 7.5). Analysis of DNA oxidized with H2O2 in the presence of Fe2+/EDTA for 30 min showed that the levels of 8-OHdG (8-hydroxy-2'-deoxyguanosine) were increased 2.7-fold, HMdU (5-hydroxymethyl-2'-deoxyuridine) 3.15-fold, and FdU (5-formyl-2'-deoxyuridine) 2.5-fold. Although the (-)-isomer of cis-dTG (cis-thymidine glycol) was enhanced 2.3 times, the (+)-isomer remained virtually unchanged. Analysis of DNA isolated from epidermal cells of mice treated in vivo with the tumor promoter TPA (12-O-tetradecanoylphorbol 13-acetate) showed 4.8-, 2.7-, and 8.7-fold increases in the levels of total cis-dTG, 8-OHdG, and HMdU, respectively, and of some unknown DNA oxidation products. These results prove applicability of the 3H-postlabeling method to the analysis of DNA (and potentially RNA) isolated from many sources, including animals and humans.
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PMID:Quantitative high-performance liquid chromatography analysis of DNA oxidized in vitro and in vivo. 188 26

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

Species variation in transcription factor IIIA (TFIIIA) was examined by comparing the abilities of TFIIIAs isolated from different Xenopus and Rana species to 1) bind rabbit anti-Xenopus laevis TFIIIA IgG, 2) specifically interact with the Xenopus borealis somatic 5S RNA gene, and 3) promote transcription of the Xenopus borealis 5S RNA gene in vitro. In immunoblot assays, Rana catesbeiana or Rana pipiens TFIIIA did not react readily with rabbit anti-Xenopus laevis TFIIIA IgG (assayed with anti-rabbit F(ab')2 fragment conjugated with alkaline phosphatase) whereas Xenopus borealis TFIIIA exhibited similar reactivity with this IgG as Xenopus laevis TFIIIA. When compared to Xenopus TFIIIAs, Rana TFIIIAs exhibited similar interactions with the 3' portion of the intragenic control region of the Xenopus 5S RNA gene (to residue +78 on the coding strand and up to and including +74 on the non-coding strand, nucleotides protected from DNase I digestion by the N-terminal half of Xenopus TFIIIA) and incomplete interactions with the remaining 5' portion of the control region (nucleotides protected from DNase I digestion by the C-terminal half of Xenopus TFIIIA). In a Xenopus laevis unfertilized egg extract, Rana catesbeiana and Rana pipiens TFIIIAs promoted transcription of the Xenopus borealis somatic 5S RNA gene less efficiently than Xenopus laevis and Xenopus borealis TFIIIAs.
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PMID:Species variation in transcription factor IIIA. 278 76

Rhizobium phage RL38JI DNA is resistant to cleavage by a variety of restriction endonucleases, and is only partially sensitive to digestion by pancreatic DNase I or by micrococcal nuclease. We have found that a mixture of DNase I, P1 nuclease, and bacterial alkaline phosphatase will quantitatively digest RL38JI DNA to deoxyribonucleosides. HPLC analysis revealed that dCyd is nearly totally absent among these digestion products, while dGuo, dAdo, and Thd are readily detected. Three additional peaks are always present; their retention properties correspond to no known modified deoxyribonucleosides. Thus it appears that dCyd is replaced in phage RL38JI DNA by as many as 3 different modified residues.
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PMID:Replacement of the deoxycytidine residues in Rhizobium bacteriophage RL38JI DNA. 298 32

The antitumor antibiotic mitomycin C is shown to form a covalent complex with calf thymus DNA under anaerobic conditions in the presence of either NADPH cytochrome c reductase/NADPH, xanthine oxidase/NADH, or the chemical reducing system H2/PtO2. Digestion of the complex with DNase I/snake venom diesterase/alkaline phosphatase yields a single mitomycin deoxyguanosine adduct as the major DNA alkylation product, identified as N2-(2'' beta,7''-diaminomitosen-1'' alpha-yl) 2'-deoxyguanosine (Structure 2). Two minor adducts, 2-5% each of the total adduct pool, are isolated and identified as the 1'' beta stereoisomer of 2 (Structure 3), and 10''-decarbamoyl-2 (Structure 7). The same results were obtained with M13 DNA and poly(dG-dC).poly(dG-dC); however, in the latter case, a minor adduct apparently possessing two deoxyguanosine and one mitomycin unit is isolated. Digestion of the covalent mitomycin-calf thymus DNA complex with nuclease P1 yields four dinucleotide adducts, all of which consist of 2 linked at its 3' end to each of the four possible 5' nucleotides (A, T, G, and C). Upon treatment of each dinucleotide adduct with snake venom diesterase/alkaline phosphatase, 2 is released along with the corresponding free nucleoside. In apparent conflict with the present results, previous reports from another laboratory have indicated that modification of calf thymus DNA by mitomycin C under conditions identical to those described here result in the isolation of three mitomycin C mononucleotide adducts possessing linkages of the drug to N2 and O6 of guanine and N6 of adenine. Evidence is shown suggesting that the latter adducts are actually three of the above four dinucleotide derivatives of 2 obtained independently by us and, thus, all of them in fact possess an identical N2-mitosenylguanine adduct moiety. Model-building studies indicate an excellent fit of the guanine N2-linked drug molecule inside the minor groove of B-DNA with no appreciable distortion of the DNA structure.
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PMID:Reaction of DNA with chemically or enzymatically activated mitomycin C: isolation and structure of the major covalent adduct. 301 44

The formation of benzo[a]pyrene (BP)-DNA adducts was studied in vitro in the presence of microsomes prepared from the isolated labyrinth zone of the rat placenta, the hematopoietic erythroblast cells of the fetal liver, the fetal liver, as well as the maternal liver. Pregnant rats received beta-naphthoflavone (beta NF; 15 mg/kg, i.p.) on day 17 gestation. One day later, placentae, fetal and maternal livers were obtained and hematopoietic erythroblast cells were separated from hepatocytes in the fetal livers. The respective microsomal fractions were incubated in the presence of calf thymus DNA, NADPH-regenerating system and [3H]BP (300 microCi) at 37 degrees C for 30 min. Following beta NF pretreatment, the levels of covalent binding (pmol/mg DNA/mg microsomal protein) for maternal liver, fetal liver, placenta and erythroblast cells were: 28.4, 2.4, 0.31 and 3.9, respectively, with the hematopoietic erythroblast cells being the most active among fetal tissue preparations. The extent of transplacental induction compared to control was greatest in the hematopoietic cells (18-fold) followed by fetal liver (16-fold) and labyrinth zone (5-fold). Further experiments characterized the BP-DNA adducts formed by induced microsomes. DNA was isolated, purified and digested sequentially with DNase I, snake venom phosphodiesterase type II and alkaline phosphatase type III. The deoxynucleoside-BP adducts were purified on a Sephadex LH-20 column and then separated on HPLC and the adducts were quantitated radiometrically. Seven distinct adducts were separated on HPLC and named A-G in order of elution. Adduct B was prominent in all preparations (22-55% total radioactivity). The adduct profile and retention time for peak B is similar to that reported for the adduct formed by microsomal activation of 9-hydroxy BP. Peak D constituted a major fraction (19%) in maternal liver profiles in comparison with the three fetal tissue preparations (8%). In subsequent experiments, peak D was shown to be derived from reaction of (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) with DNA. Peak C was unique to erythroblast cell and labyrinth profiles, while peak G was specific for maternal liver and fetal liver profiles. These results demonstrate that fetal liver and its hematopoietic cells are significant sites of BP bioactivation which may contribute to the fetal toxicity of polyaromatic hydrocarbons.
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PMID:Formation of benzo[a]pyrene-DNA adducts by microsomal enzymes: comparison of maternal and fetal liver, fetal hematopoietic cells and placenta. 356 91


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