A new method for enzymatic preparation of isopentenyladenine-type and trans-zeatin-type cytokinins with radioisotope-labeling.
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.
Takei
Kentaro
K
Plant Science Center, RIKEN (The Institute of Physical and Chemical Research), 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan.
Dekishima
Yasumasa
Y
Eguchi
Tadashi
T
Yamaya
Tomoyuki
T
Sakakibara
Hitoshi
H
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D002417:Cattle; D003583:Cytokinins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005786:Gene Expression Regulation; D007541:Isopentenyladenosine; D015394:Molecular Structure; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism; D014316:Tritium; D015026:Zeatin / Q000031:analogs & derivatives / Q000378:metabolism*
D003583:Cytokinins; D014316:Tritium; C001478:N(6)-(delta(2)-isopentenyl)adenine; D007541:Isopentenyladenosine; D015026:Zeatin; D011683:Purine-Nucleoside Phosphorylase; D019883:Alkyl and Aryl Transferases; C024274:adenylate isopentenyltransferase; D000469:Alkaline Phosphatase; D000225:Adenine