Cytokinin oxidase from wheat: partial purification and general properties

Abstract

As part of the study of the possible role(s) of CBF-1, a cytokinin-binding protein abundant in wheat embryo, a cytokinin oxidase was found in wheat (Triticum aestivum L.) germ and partially purified by conventional purification techniques and high performance chromatofocusing. This preparation catalyzes conversion of N(6)-(Delta(2)-isopentenyl)adenosine to adenosine at a V(max) of 0.4 nanomol per milligram protein per minute at 30 degrees C and pH 7.5, the K(m) being 0.3 micromolar. This high affinity and the apparent molecular weight of 40,000 estimated by high performance gel permeation on a Spherogel TSK-3000 SW column indicate that this enzyme is different from other cytokinin oxidases previously reported. Oxygen is required for the reaction, as for other cytokinin oxidases already described. N(6)-(Delta(2)-isopentenyl)adenine and zeatin riboside are also degraded, but N(6)-(Delta(2)-isopentenyl)adenosine-5'-monophosphate is apparently not a substrate. Benzyladenine is degraded, but to a small extent, and it inhibits slightly the degradation of N(6)-(Delta(2)-isopentenyl)adenosine. The degradation of N(6)-(Delta(2)-isopentenyl)adenosine is strongly inhibited by diphenylurea and its highly active derivative N-(2-chloro-4-pyridyl)-N'-phenylurea.