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)

DNA was subjected to bisulfite-catalyzed transamination at the N4 sites of its cytosine residues with 1,8-diaminooctane (DAO). The product, DNA-DAO, was non-specifically degraded with a cloned staphylococcal nuclease (Nase). The products from the Nase digestion were determined by high-performance liquid chromatography (HPLC) to define the extent of reaction with DAO. Mostly, nucleoside 3'-monophosphates were obtained, along with four Nase-resistant dinucleotides: TpdGp, dApdGp, TpdCp-DAO and dApdCp-DAO. The addition of spleen phosphodiesterase II gave a faster hydrolysis and left no dinucleotides. A mixture of Nase, snake venom phosphodiesterase I and alkaline phosphatase gave a fast hydrolysis as well but two dinucleotides, apparently TpdC-DAO and dApdC-DAO, persisted. Further modification of the diaminooctyl side chains with fluorescein isothiocyanate or biotin N-hydroxysuccinimide ester was similarly investigated. Interestingly, derivatization of the DAO side chain with biotin eliminates the resistance of TpdCp-DAO and dApdCp-DAO to Nase digestion. This work provides some guidelines for using Nase, alone or with other nucleases, along with HPLC, for characterizing alkyldiamine DNA products, and should be similarly useful for studying other modifications of DNA.
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PMID:Staphylococcal nuclease high-performance liquid chromatographic characterization of diaminooctane-modified DNA and its biotin and fluorescein derivatives. 284 10

o-Phosphotyrosyl glutamine synthetase (P-GS) was isolated from highly adenylated glutamine synthetase (AMP-GS) purified from Mycobacterium phlei, by treatment with micrococcal nuclease. The physical characteristics of P-GS were quite similar to those of AMP-GS except for the UV-absorption spectrum. In either Mg2+- or Mn2+-dependent biosynthetic reactions, the kinetic properties, such as optimum pH, Vmax, and apparent Km for each of three substrates of P-GS, were found to be in good agreement with those of AMP-GS. The biosynthetic activity of P-GS was markedly increased after treatment with alkaline phosphatase similarly as in the deadenylylation of AMP-GS by snake venom phosphodiesterase treatment. These results revealed that repression of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue, without recourse to adenylylation.
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PMID:Regulation of glutamine synthetase activity by phosphorylation instead of by adenylylation. 290 54

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins. 324 13

Conversion of absorbance unit into molar amount was tried as to deoxydinucleotide. In order to achieve this trial, a reversed-phase high-performance liquid chromatographic method with a linear gradient mode was established for separation and quantification of deoxymononucleotides and deoxynucleosides. Selecting 2'-deoxycytidylyl-(3'-5')-thymidylic (5') acid as an example of deoxydinucleotides, this compound dissolved in water was estimated in absorbance unit at 260 nm, and it was treated by alkaline phosphatase and furthermore by snake venom phosphodiesterase. Based on quantification of the cleaved nucleic acid constituents including 2'-deoxycytidine 5'-monophosphoric acid and thymidine by the above method, the molar amount per 1.000 absorbance unit at 260 nm in the deoxydinucleotide was determined. Thus, the conversion of absorbance unit into molar amount is considered to be applicable to other deoxyoligonucleotides.
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PMID:Determination of deoxymononucleotides and deoxynucleosides as a tool for toxicological examination--a trial for conversion of absorbance unit into molar amount in deoxydinucleotide. 338 9

Porfiromycin was reductively metabolized by NADPH cytochrome P-450 reductase and xanthine oxidase under anaerobic conditions. The production of metabolites varied with the pH and the contents of the reaction buffer. In Tris buffer, two major metabolites were produced at pH 7.5 and above, whereas one major metabolite was produced at pH 6.5. The three major metabolites were separated and isolated by HPLC. Identification by californium-252 plasma desorption mass spectrometry showed that the two major metabolites from pH 7.5 were (trans) and (cis)-forms of 7-amino-1-hydroxyl-2-methylaminomitosene and the major metabolite from pH 6.5 was 7-amino-2-methylaminomitosene. All three major metabolites showed substitutions at the C-1 position. DNA was alkylated readily by enzyme-activated porfiromycin. Digestion of porfiromycin-alkylated DNA by DNase, snake venom phosphodiesterase, and alkaline phosphatase resulted in an insoluble nuclease-resistant fraction and a soluble fraction. The nuclease-resistant fraction reflected a high content of cross-linked adducts. Upon HPLC analysis, the solubilized fraction contained two monofunctionally linked porfiromycin adducts and a possibly cross-linked dinucleotide. The major adduct was isolated by HPLC and identified by NMR, as N2-(2'-deoxyguanosyl)-7-amino-2-methylaminomitosene. The N2 position of deoxyguanosine appeared as the major monofunctional alkylating site for DNA alkylation by porfiromycin. Thus, mitomycin C and porfiromycin (which differs from mitomycin C only by the addition of a methyl group to the aziridine nitrogen) share the same enzymatic activating mechanism that leads to the formation of the same types of metabolites and the same specificity of DNA alkylation.
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PMID:Metabolites and DNA adduct formation from flavoenzyme-activated porfiromycin. 341 25

A novel nucleotide, Ypp5'A2'p, has been purified through perchloric acid extraction of rat liver followed by DEAE-cellulose and ion pair high pressure liquid chromatographies. Y stands for an unknown compound, probably a nucleoside, whose sugar moiety is different to beta-D (deoxy) ribose. Treatment of Ypp5'A2'p with snake venom phosphodiesterase renders Yp and adenosine 2',5'-bisphosphate (pAp). After elimination of the terminal phosphate with alkaline phosphatase, the resulting nucleotide (Ypp5'A) yielded Yp and 5'-AMP when hydrolyzed by the phosphodiesterase. The following ultraviolet absorption spectral characteristics were determined at pH 7: Ypp5'A2'p (lambda max = 265 nm; A250/A260 = 0.76; A280/A260 = 0.79); Yp (lambda max = 279 nm; A250/A260 = 0.70; A280/A260 = 1.70). The molar extinction coefficient found for Yp at 280 nm was 20.6 x 10(3) M-1 cm-1.
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PMID:Rat liver contains a novel nucleotide, Ypp5'A2'p, related to adenosine 2',5'-bisphosphate. 343 33

Endogenous oligonucleotides were found in trichloroacetic acid extracts of hamster lung fibroblasts and Tetrahymena cells. Peaks of radioactivity that eluted with retention times similar to oligonucleotide markers (5- to 50-mer) were found by HPLC in cells labeled briefly with 32Pi. Only minute amounts of UV-absorbing material were detected, consistent with a rapid turnover of phosphate groups. The 32P-labeled material also migrated as oligonucleotides on 20% polyacrylamide gels; it was not hydrolyzed by alkaline phosphatase but was digested by snake venom phosphodiesterase, S1 nuclease, and pancreatic RNase and was phosphorylated by T4 polynucleotide kinase. The 32P-labeled material isolated by HPLC was alkali labile and the hydrolyzate ran as nucleotides on paper chromatography. It is concluded that the oligonucleotides are mainly oligoribonucleotides, but it is possible that oligodeoxynucleotides are also present.
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PMID:Oligonucleotides with rapid turnover of the phosphate groups occur endogenously in eukaryotic cells. 347 Jul 67

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

An enzyme that converts [3H, 32P]ATP, with a 3H:32P ratio of 1:1, to oligoadenylates with the same 3H:32P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of 3H:32P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities. 399 78

13C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% 13C-labelled carbon dioxide. The 13C enriched DNA was quantitatively hydrolysed with DNase I, snake venom phosphodiesterase I and alkaline phosphatase of intestinal mucosa. The resulting deoxyribonucleosides were separated by preparative reversed-phase high pressure liquid chromatography in 60 minutes with detection by ultraviolet absorption at 254 nm. The final products were obtained in milligram quantities in high purity and in high yield. The 1H resonances of the base and sugar protons of these deoxyribonucleosides appear as well resolved multiplets in the 600 MHz NMR spectrum, due to the extensive 1H-13C couplings. Similarly, the 13C resonances of these deoxyribonucleosides appear as multiplets in the 75.5 MHz 13C NMR spectrum, due to 13C-13C couplings. The 1H-13C and 13C-13C coupling constants were also measured and tabulated. The isotopic enrichment of 13C these deoxyribonucleosides was obtained by integration of the 1H and/or 13C NMR spectra. It was found that the enrichment varied from carbon to carbon and species to species in the range of 70-89%, suggesting differential uptake and assimilation of 90% 13CO2 during metabolism pathways. This protocol provides experimentally useful quantities of 13C-enriched deoxyribonucleosides, which may be incorporated into site-specifically labeled oligonucleotides by chemical synthesis.
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PMID:Isolation and purification of deoxyribonucleosides from 90% 13C-enriched DNA of algal cells and their characterization by 1H and 13C NMR. 400 Sep 54


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