Graduate Thesis Or Dissertation
 

Cytokinin oxidase activity from Phaseolus vulgaris L. cv. Great Northern callus tissues

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  • Cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv. Great Northern has been examined using an assay based on the oxidation of radioactively labeled N⁶ -(Δ² -isopentenyl) adenine (i⁶Ade) to adenine (Ade). Conditions for the quantitative extraction and assay of the enzyme have been established. The substrate specificity of the enzyme appears similar to that reported for cytokinin oxidase preparations from other plant sources. Solutions of exogenous cytokinins applied directly to the surface of Great Northern callus tissues induced relatively rapid (in less than an hour) increases in cytokinin oxidase activity. The cytokinin-induced increase in cytokinin oxidase activity appears to require RNA and protein synthesis. All cytokinin-active compounds tested, including substrates and non-substrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokininactive urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Cu⁺² to cytokinin oxidase assay mixtures containing imidazole buffer enhanced the in vitro activity of the enzyme more than 20-fold. The effect was enzyme dependent and specific for copper and the cytokinin oxidase catalyzed reaction. In the presence of copper and imidazole, the degradation of i⁶Ade to Ade catalyzed by cytokinin oxidase was observed to proceed under anaerobic conditions. This result suggests that the copper-imidazole complex is substituting for oxygen as an electron accepter in the cytokinin oxidase reaction. The chromatographic properties of the cytokinin oxidase activity have also been investigated. Most of the cytokinin oxidase activity extracted from callus tissues bound to concanavalin A-Sepharose 4B and was specifically eluted with methyl-mannose. This result suggests that the enzyme is a glycoprotein. DEAE-cellulose chromatography resolved the cytokinin oxidase activity into two peaks. The major fraction, which comprised 85% to 90% of the cytokinin oxidase activity extracted from the callus tissues, bound to concanavalin A-Sepharose 4B. The minor peak of cytokinin oxidase activity did not. These results provide evidence for the presence of multiple forms of cytokinin oxidase, but the possibility of artifacts generated during enzyme preparation cannot yet be excluded.
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