Purification and cDNA cloning of cytokinin-specific binding protein from mung bean (Vigna radiata)

Authors


  • Correspondence to Y. Hashimoto, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan 113-0032

  • Fax: +81 3 5684 8629.

  • E-mail:hashimot@imcbns.iam.u-tokyo.ac.jp

  • Abbreviations. 4PU, N-phenyl-N′-(4-pyridyl)urea; 4PU30, N-(2-chloro-4-pyridyl)-N′-phenylurea; RT-PCR, reverse transcription-polymerase chain reaction; GST, glutathione S-transferase; WCE, whole cell extract; DPU, N,N′-diphenylurea.

  • Note. The novel nucleotide sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases and are available under accession numbers AB012218, AB012219 and AB012220. Y. Fujimoto and R. Nagata contributed equally to this publication.

Abstract

Synthetic urea derivatives such as N-phenyl-N′-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N′-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we previously established the presence of a cytokinin-specific binding protein (CSBP) of high affinity (Ka for 4PU30 = 4×1010 M−1) in the soluble fraction of etiolated mung bean seedlings [Nagata, R., Kawachi, E., Hashimoto, Y. & Shudo, K. (1993) Biochem. Biophys. Res. Commun. 191, 543−549]. In this report, we purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel liganded with 4PU. We determined partial amino acid sequences of CSBP and isolated its cDNA by reverse-transcription (RT) PCR. The cDNA encoded a protein with a calculated molecular mass of 17 kDa. A data base homology search revealed that CSBP is a novel member of a major pollen allergen/pathogenesis-related protein family. Recombinant CSBP was expressed in Escherichia coli and was confirmed to bind specifically to cytokinins.

Ancillary