Rate enhancement of cytokinin oxidase/dehydrogenase using 2,6-dichloroindophenol as an electron acceptor |
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Authors: | James G Laskey Paige Patterson Kristin Bilyeu Roy O Morris |
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Institution: | (1) Department of Biochemistry, University of Missouri, Columbia, USA;(2) Plant Genetics Research Unit, USDA-ARS, Columbia, USA |
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Abstract: | Cytokinin oxidase/dehydrogenase degrades cytokinins by dehydrogenating the N6-C1 bond of cytokinins. The resulting imine is then hydrolyzed. For example, isopentenyl-adenine is cleaved into 3-methyl-2-butenal (isopentenyl-aldehyde) and adenine . The reducing equivalents from dehydrogenation are transferred to an unknown sink, in vivo. It has been hypothesized that the enzyme requires oxygen , possibly resulting in the formation of hydrogen peroxide. 2,6-dichloroindophenol (DCPIP) can function as an acceptor of reducing equivalents for in vitro cytokinin oxidase/dehydrogenase reactions. For the predominant cytokinin oxidase/dehydrogenase in maize, ZmCKX1, the addition of DCPIP to in vitro reactions increases the reaction rate to nearly 4000-fold faster than the oxygen-dependent rate. Further, the change in absorbance of DCPIP at 600 nm, as it is reduced, forms the basis for an assay suitable for following biochemical purification of cytokinin oxidase/dehydrogenases , detailed kinetic studies , and rapid measurement of cytokinin oxidase/dehydrogenase activity in large numbers of samples. |
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Keywords: | Assay Cytokinin Cytokinin Oxidase/dehydrogenase DCPIP |
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