A. Rose et al., PURIFICATION AND CHARACTERIZATION OF GLYCOSYLTRANSFERASES INVOLVED INANTHOCYANIN BIOSYNTHESIS IN CELL-SUSPENSION CULTURES OF DAUCUS-CAROTAL, Planta, 198(3), 1996, pp. 397-403
The major anthocyanins accumulated by an Afghan cultivar of Daucus car
ota L. are cyanidin 3-(xylosylglucosylgalactosides) acylated with sina
pic or ferulic acid. The formation of the branched triglycoside presen
t as a common structural element requires an ordered sequence of glyco
sylation events. Two of these enzymic glycosylation reactions have bee
n detected in protein preparations from carrot cell-suspension culture
s. The first step is a galactosyl transfer catalyzed by UDP-galactose:
cyanidin galactosyltransferase (CGT) resulting in cyanidin 3-galactos
ide. The putative second step is the formation of cyanidin 3-(xylosylg
alactoside) catalyzed by UDP-xylose: cyanidin 3-galactoside xylosyltra
nsferase (CGXT). Both enzyme activities were characterized from crude
protein preparations. The CGT was purified 526-fold from the cytosolic
fraction of UV-irradiated cell cultures by ion-exchange chromatograph
y on diethylaminoethyl (DEAE)-Sephacel, affinity chromatography on Blu
e Sepharose CL-6B, gel permeation chromatography on Sephadex G-75 and
elution from the gel matrix after non-dissociating PAGE. Its molecular
mass was estimated by SDS-PAGE and by calibrated gel permeation chrom
atography on Sephadex G-75. In both cases a molecular mass of 52 kDa w
as determined, indicating that the native protein is a monomer of 52 k
Da. The galactosyl transfer and the xylosyl transfer are presumed to b
e catalyzed by separate enzymes.