81 アズキ GBP とアズキ GA 2-oxidase 酵素の性状比較(2)

概要

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We have reported the purification of a gibberellin-binding protein (GBP) from adzuki bean seedlings. To purify the GBP further, we examined various conditions for a cation exchange chromatography (CEC) with the SMART system to obtain high recovery of GA-binding activity. The tandem purification with CEC was very effective. SDS-PAGE for the active fraction and its neighbors was performed and showed that a bunch of proteins with molecular masses of 37-40 kDa were always existed in the active fractions. The molecular mass is very similar to that of GA 2-oxidases in adzuki bean seedlings. To elucidate whether the GBP was identical to 2-oxidases, we took two approaches; (i) the immunological detection of 2-oxidase in the active fraction and (ii) the sequential analysis for the proteins in the active fraction. We checked the expression level of 2-oxidase mRNAs first, using probes prepared from the cDNAs of adzuki 2-oxidase. Then we prepared anti-2-oxidase antisera using a mixture of two recombinants whose expression levels were predominant to other homologs in northern blotting. Western blotting analysis is proceeding. We processed over 100 kilograms of materials to purify GBP and are analyzing the N-terminus sequences of the proteins in the active fraction, especially those with molecular masses of 37-40 kDa.
植物化学調節学会の論文
2002-10-31