Abstract
We have reported the characteristics and partial purification of a gibberellin-binding protein (GBP) in cytosolic fraction from azuki bean seedlings. It's binding activity for GA_4 was detected with an unequilibrated gel-filtration technique and a tritiated GA_4 derivative. The GBP showed a high affinity to so-called 'active GAs' while it showed a low affinity to biologically inactive GAs. Here we report a further purification of the GBP where a reversed phase chromatography was decisive in its isolation. After an extraction of buffer-soluble proteins and a precipitation by salting-out, the GBP was efeiciently purified by successive perfusion chromatographies using three ion exchange columns. The fraction showing the GA-specific binding activity was obtained after the binding assay. The active fraction was subjected to a reversed phase perfusion chromatography and gave two active fractions, which were collected and characterized respectively. Their molecular masses were estimated to be around 30 kDa by GPC. The both actiive fractions showed the same K_d value (3×10^<-10>) to GA_4 in their scatchard plots analysis and over hundred kilograms of seedlings would be required for the isolation of each. The two active fractions were subjected ot GPC, and then to a reversed phase HPLC of SMART system. The use of eluent containing TFA for a Reversed phase HPLC specifically delayed the t_Rs of both GBPs sompared to t_Rs of major contaminants in the active fractions, and most of contaminants were effectively removed. We are routinely collecting the purified fractions enough for the N-terminus sequencing.
