- Synthesis of [15N4] purine labeled cytokinin glycosides derived from zeatins and topolins with 9-β-d, 7-β-d-glucopyranosyl, or 9-β-d-ribofuranosyl group
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Synthesis of [15N4] purine labeled cytokinine glycosides derived from zeatins and topolins containing a 9-β-d, 7-β-d-glucopyranosyl, or 9-β-d-ribofuranosyl group is described. These N6-substituted adenine derivatives are i
- Tranová, Lenka,Bu?ek, Jan,Zatloukal, Marek,Canka?, Petr,Styskala, Jakub
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p. 118 - 125
(2019/01/24)
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- A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with specificity for cytokinins contributes to the duration of tuber endodormancy
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StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase) catalyses the interconversion of the N9-riboside form of the plant hormone CK (cytokinin), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as a CK-binding activity in extracts of tuberizing potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide and motifs for purine nucleoside phosphorylase activity. The expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted into ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted into their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.
- Bromley, Jennifer R.,Warnes, Barbara J.,Newell, Christine A.,Thomson, Jamie C.P.,James, Celia M.,Turnbull, Colin G.N.,Hanke, David E.
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p. 225 - 237
(2014/03/21)
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- Peroxide-shunt substrate-specificity for the Salmonella typhimurium O 2-dependent tRNA modifying monooxygenase (MiaE)
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Post-transcriptional modifications of tRNA are made to structurally diversify tRNA. These modifications alter noncovalent interactions within the ribosomal machinery, resulting in phenotypic changes related to cell metabolism, growth, and virulence. MiaE is a carboxylate bridged, nonheme diiron monooxygenase, which catalyzes the O2-dependent hydroxylation of a hypermodified-tRNA nucleoside at position 37 (2-methylthio-N6- isopentenyl-adenosine(37)-tRNA) [designated ms2i6A 37]. In this work, recombinant MiaE was cloned from Salmonella typhimurium, purified to homogeneity, and characterized by UV-visible and dual-mode X-band EPR spectroscopy for comparison to other nonheme diiron enzymes. Additionally, three nucleoside substrate-surrogates (i6A, Cl2i6A, and ms2i6A) and their corresponding hydroxylated products (io6A, Cl2io 6A, and ms2io6A) were synthesized to investigate the chemo- and stereospecificity of this enzyme. In the absence of the native electron transport chain, the peroxide-shunt was utilized to monitor the rate of substrate hydroxylation. Remarkably, regardless of the substrate (i6A, Cl2i6A, and ms2i6A) used in peroxide-shunt assays, hydroxylation of the terminal isopentenyl-C4-position was observed with >97% E-stereoselectivity. No other nonspecific hydroxylation products were observed in enzymatic assays. Steady-state kinetic experiments also demonstrate that the initial rate of MiaE hydroxylation is highly influenced by the substituent at the C2-position of the nucleoside base (v0/[E] for ms2i6A > i 6A > Cl2i6A). Indeed, the >3-fold rate enhancement exhibited by MiaE for the hydroxylation of the free ms 2i6A nucleoside relative to i6A is consistent with previous whole cell assays reporting the ms2io6A and io6A product distribution within native tRNA-substrates. This observation suggests that the nucleoside C2-substituent is a key point of interaction regulating MiaE substrate specificity.
- Corder, Andra L.,Subedi, Bishnu P.,Zhang, Siai,Dark, Amanda M.,Foss Jr., Frank W.,Pierce, Brad S.
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p. 6182 - 6196
(2013/10/01)
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- Efficiency of different methods of extraction and purification of cytokinins
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The increasing use of advanced methods, such as mass spectrometry, for the determination of cytokinins has raised special requirements for the extraction and purification of this class of plant hormones. Extraction of Arabidopsis thaliana plants with three different solvents, [80% (v/v) MeOH, Bieleski's MCF-7, and modified Bieleski's] provided similar yields of most analyzed cytokinins determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). However, the extraction with a modified Bieleski's solvent (MeOH-HCO2H-H2O [15:1:4, v/v/v]) gave the highest responses of deuterated cytokinins (used as test compounds) in plant extracts as compared to the responses of pure deuterated standards (relative internal standard response, RISR). Purification of cytokinins using Oasis MCX sorbent with reversed-phase and cation-exchange characteristics, in comparison to the DEAE Sephadex RP-C18 method, provided higher levels of zeatin riboside monophosphate and similar levels of cytokinin bases, ribosides and glucosides. Using this method the content of UV-absorbing contaminates was decreased by about 90% and the RISR values of all tested cytokinin standards but riboside monophosphates were increased about two-fold. The former method provided preparations more suitable for HPLC/MS/MS analysis with respect to simplicity and sample purity.
- Hoyerova, Klara,Gaudinova, Alena,Malbeck, Jiri,Dobrev, Petre I.,Kocabek, Tomas,Solcova, Blanka,Travnickova, Alena,Kaminek, Miroslav
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p. 1151 - 1159
(2008/02/10)
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- SIMPLE, INEXPENSIVE ROUTES TO E AND Z ZEATINE RIBOSIDES AND DERIVATIVES USEFUL FOR IMMUNOASSAY.
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Allylic oxidation of 6-N-(3,3-dimethylallyl)adenosine 1 gave trans (E) zeatine riboside 3, which was isomerized to the cis (Z) isomer 5 by u.v. irradiation.A method for the regiospecific 3'-O-succinoylation of these nucleosides is given.
- David, Serge,Sennyey, Gerard de,Sotta, Bruno
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p. 1817 - 1820
(2007/10/02)
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