Cytokinin-specific Binding Protein Research Articles

Molecular identification of cytokinin-specific binding protein

Synthetic phenylurea 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 found the presence of a cytokinin-specific binding protein (CSBP) with high affinity for 4PU30 (Ka for 4PU30 = 4 x 10(10) M-1) in the soluble fraction of etiolated mung bean seedlings. We purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel ligated with 4PU. Analysis of its cDNA revealed that CSBP was a novel member of a major pollen allergen/pathogenesis-related protein family with a calculated molecular weight of 17 kDa. Recombinant CSBP was expressed in Escherichia coli was confirmed to bind specifically to cytokinins.

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

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 = 4x10(10) 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.

Cytokinin-Specific Binding-Protein in Etiolated Mung Bean Seedlings

We have found that a synthetic urea derivative, N-(2-chloro-4-pyridyl)- N′-phenylurea (4PU30, forchlorfenuron), has even higher cytokinin activity than N 6-benzyladenine (BA). Using [ 3H]PU30 (2.83 TBq/mmol) as a probe, we confirmed chromatographically the presence of a high-affinity cytokinin-specific binding protein (CSBP) in whole-cell extract from etiolated mung bean seedlings. The apparent molecular weight of CSBP was estimated to be 21 kDa. The association constants (K a′s) of CSBP for 4PU30 and BA were calculated to be 4 × 10 10) M −1 and 3 × 10 9 M −1, respectively. Various active cytokinins showed mutually competitive binding to CSBP, and their affinities for CSBP corresponded well to their cytokinin activities at the tissue level.