Detection and Characterization of UDP-Glucose:Flavonoid O-Glucosyltransferases in the Leaves of Prunus×yedoensisMatsum

摘要

A novelO-glucosyltransferase (I4′GT) which catalyzes the transfer of D-glucose from UDP-D-glucose to position 4'of prunetin (4′,5-dihydroxyl-7-methoxyisoflavone) was isolated from the leaves ofPrunus×yedoensisMatsum. and purified 66-fold by precipitation with ammonium sulfate and chromatography on DEAE-cellulose. UDP-glucose:flavonol 3-O-glucosyltransferase (F3GT) was also isolated and purified 50-fold in the same manner. The molecular weights of both I4′GT and F3GT were estimated by elution from a column of Sephadex G-100 to be about 51,000 Da. The pH optima for I4′GT and F3GT activities were 8.0 and 7.5, respectively. The specificities of I4′GT and F3GT for the sugar donor were quite strict, and only UDP-glucose could serve as glucosyl donor, both ADP-D-glucose and GDP-D-glucose being ineffective. The apparentKmvalues for UDP-glucose and prunetin were 10.0μMand 1.20μM, respectively, for I4′GT. TheKmvalues for UDP-glucose and quercetin were 9.8μMand 1.21μM, respectively, for F3GT. The activities of both I4′GT and F3GT were stimulated by 1 mM Mg*+and strongly inhibited by 1 mM Cu2+, 1 mM Zn2+and various reagents that react with