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We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine
(iP), trans-zeatin (tZ), and their ribosides. The method is based on the three enzyme activities of an adenylate isopentenyltransferase
(IPT; EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli. The A. thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate as isoprenoid donors. The dual specificity of the substrates enabled us to produce iP-type and tZ-type
cytokinins separately in the same system simply by switching the substrates. Our method affords a much higher yield of the
labeled products than the chemical reaction methods previously used. These labeled compounds will be useful tools for cytokinin
research, such as receptor–ligand assays and cell metabolism studies. 相似文献
2.
Recent progress in the study of cytokinin metabolism in plants indicates that quantitative analysis of cytokinin nucleotides
is essential for elucidation of early steps of the biosynthetic pathway. However, traditional procedures for purification
and quantification of cytokinin cannot discriminate the various nucleotides. We describe here a method for separation and
determination of cytokinin nucleotides through a series of anion-exchange column chromatography steps followed by liquid chromatography-mass
spectrometry. This method enabled us to analyze the amount of each species of cytokinin nucleotide in plant tissues. 相似文献
3.
The phloem sap of Ricinus seedlings was analyzed for cytokinins and the concentration was compared with that in cotyledons and xylem sap. The dominant cytokinin in the phloem sap was isopentenyladenine (70 nM) when the endosperm was attached to the cotyledons; zeatin, dihydrozeatin and cytokinin-ribosides were present at relatively low concentrations (1–2 nM). Removal of the endosperm and incubation of the cotyledons in buffer led to a sharp decrease in the level of isopentenyladenine in the phloem sap, down to the value for zeatin, namely 1–2 nM. Similar low cytokinin concentrations were found in the xylem sap, too, whereas in the cotyledons the cytokinin content was at least 10-fold higher. Incubation of the cotyledons with various cytokinins (isopentenyladenine, zeatin and their ribosides) led to an increase of each of the applied cytokinins in the phloem sap, including also the metabolically closely related cytokinins. Zeatin was especially well loaded. It is concluded that the phloem translocates most free bases and ribosides of the various cytokinin species, if they are offered to the phloem. The data also show that the cytokinin levels in the phloem, which may be far higher than in the xylem, are subject to strong fluctuations depending on the physiological situation.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137). The experimental assistance by P. Geigenberger and the help in cytokinin analysis by Dr. A. Fußeder, Dr. B. Wagner, W. Peters (all Bayreuth) and by Prof. E. Weiler (Bochum) is gratefully acknowledged. Also the constructive discussions with Profs. E. Weiler (Bochum) and E. Beck (Bayreuth) are much appreciated. 相似文献
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