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 220.127.116.11) from Arabidopsis thaliana, an alkaline phosphatase (EC 18.104.22.168) from calf intestine, and a purine-nucleoside phosphorylase (EC 22.214.171.124) 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.
- Adenylate isopentenyltransferase
- Purine-nucleoside phosphorylase
- Radioisotope labeling
ASJC Scopus subject areas
- Plant Science