Cytokinin Oxidase/Cytokinin Dehydrogenase Assay: Optimized Procedures and Applications

Spectrophotometric methods for determining the activity of cytokinin oxidase/cytokinin dehydrogenase (EC 1.5.99.12) were developed and optimized. A sensitive end-point method based on a combination of the electron acceptor 2,6-dichlorophenolindophenol and Schiff base formation of the reaction product with 4-aminophenol under acidic conditions can be applied to crude cell and tissue extracts. The assay was also adapted for other substrates than N6-(2-isopentenyl)adenine, such as zeatin and the aromatic cytokinins, although an enzyme which degrades the latter compounds has not yet been identified. The second novel method is an initial rate method based on the coupled redox reaction of phenazine methosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide resulting in the formation of a formazan dye. This method can be used for kinetic studies with purified enzyme and is entirely substrate independent.