Cytokinin-binding proteins from tobacco callus share homology with osmotin-like protein and an endochitinase.
To study the signal transduction of cytokinins, we characterized cytokinin-binding proteins (CBPs) isolated from tobacco callus Nicotiana tabacum. Two high-affinity CBPs, CBP1 and CBP2, were isolated from the soluble fraction of tobacco callus BY-2 cells by anion exchange chromatography on a DEAE-cellulose column and affinity chromatography on a benzyladenine (BA)-linked Sepharose 4B column. Cytokinin-binding activity was determined by the equilibrium dialysis method. The degree of purification of CBP1 and CBP2 was 270 and 600-fold, respectively. These proteins had molecular masses of 34 kDa and 26 kDa, and to bind benzyladenine (BA) with dissociation constants (Kd) of 8.9 x 10(-6) M and 1.1 x 10(-6) M, respectively. Binding of BA to CBP2 was inhibited by zeatin and kinetin but not by adenine, adenosine, ATP or IAA. The optimum pH for binding of BA to CBP1 and CBP2 was approximately pH 6.5 and 7.5, respectively. CBP1 showed significant homology (90%) with endochitinase and CBP2 with osmotin-like protein (OLP). These findings and the results of immunoblotting analysis and cytokinin-binding assay of recombinant OLP indicated that CBP2 is OLP, a stress protein.
Kobayashi
K
K
Laboratory of Life Sciences, Tokyo Gakugei University, Japan.
Fukuda
M
M
Igarashi
D
D
Sunaoshi
M
M
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins; D002352:Carrier Proteins / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D005591:Chemical Fractionation; D002688:Chitinases / Q000378:metabolism*; D003583:Cytokinins / Q000378:metabolism*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism*; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014026:Nicotiana / Q000378:metabolism
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D003583:Cytokinins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C073139:osmotin protein, Nicotiana tabacum; D002688:Chitinases