PURIFICATION AND CHARACTERIZATION OF GLYCOSYLTRANSFERASES INVOLVED INANTHOCYANIN BIOSYNTHESIS IN CELL-SUSPENSION CULTURES OF DAUCUS-CAROTAL

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.