Isolation of cytokinin binding protein from tobacco leaves by bioaffinity chromatography and its partial characterization.
Cytokinin binding protein was isolated and purified from tobacco leaves by bioaffinity chromatography on a Sepharose column on which benzyladenine (BA), synthetic cytokinin, had been introduced as an affinity ligand by the cyanogen bromide method. The purified protein bound specifically to cytokinins; its binding was inhibited remarkably by the addition of BA and kinetin and slightly by adenine, but not by adenosine in vitro. The dissociation constant, Kd, of the protein-BA complex was about  4 x 10(-5)M. The profiles of SDS polyacrylamide gel electrophoresis and gel filtration indicate that the protein consisted of a single polypeptide chain. Amino acid analysis showed that the protein contained 4 basic, 6 acidic, and 25 neutral amino acids but lacked tryptophan. The molecular weight of the protein was determined to be about 4,000 to 5,000 daltons by gel filtration, SDS polyacrylamide gel electrophoresis and amino acid analysis. The coupling conditions of BA to Sepharose by the cyanogen bromide method are described and discussed.
D000225:Adenine; D000241:Adenosine; D000596:Amino Acids / Q000032:analysis; D001593:Benzyl Compounds; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003583:Cytokinins / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010937:Plant Growth Regulators / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D010947:Plants, Toxic; D013056:Spectrophotometry, Ultraviolet; D014026:Nicotiana / Q000378:metabolism
D000596:Amino Acids; D001593:Benzyl Compounds; D002352:Carrier Proteins; D003583:Cytokinins; D010937:Plant Growth Regulators; D000225:Adenine; D000241:Adenosine