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.
Frébort
Ivo
I
Department of Biochemistry, Faculty of Science, Palacký University, Slechtitelů 11, Olomouc, Czech Republic. frebot@risc.upol.cz
Sebela
Marek
M
Galuszka
Petr
P
Werner
Tomás
T
Schmülling
Thomas
T
Pec
Pavel
P
D015086:2,6-Dichloroindophenol; D000627:Aminophenols / Q000378:metabolism; D003583:Cytokinins / Q000378:metabolism*; D004583:Electrons; D005562:Formazans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008773:Methylphenazonium Methosulfate; D010088:Oxidoreductases / Q000032:analysis / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D012545:Schiff Bases / Q000378:metabolism; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods*; D014908:Triticum / Q000201:enzymology; D003313:Zea mays / Q000201:enzymology
D000627:Aminophenols; D003583:Cytokinins; D005562:Formazans; D012545:Schiff Bases; D008773:Methylphenazonium Methosulfate; D015086:2,6-Dichloroindophenol; D010088:Oxidoreductases; C019984:cytokinin oxidase; C026729:4-aminophenol