title: Purification and characterization of glycosyltransferases involved in anthocyanin biosynthesis in cell-suspension cultures of Daucus carota L.
abstract: The major anthocyanins accumulated by an Afghan cultivar of Daucus carota L. are cyanidin 3-(xylosylglucosylgalactosides) acylated with sinapic or ferulic acid. The formation of the branched triglycoside present as a common structural element requires an ordered sequence of glycosylation events. Two of these enzymic glycosylation reactions have been detected in protein preparations from carrot cell-suspension cultures. The first step is a galactosyl transfer catalyzed by UDP-galactose: cyanidin galactosyltransferase (CGT) resulting in cyanidin 3-galactoside. The putative second step is the formation of cyanidin 3-(xylosylgalactoside) catalyzed by UDP-xylose: cyanidin 3-galactoside xylosyltransferase (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 chromatography on diethylaminoethyl (DEAE)-Sephacel, affinity chromatography on Blue 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 chromatography on Sephadex G-75. In both cases a molecular mass of 52 kDa was determined, indicating that the native protein is a monomer of 52 kDa. The galactosyl transfer and the xylosyl transfer are presumed to be catalyzed by separate enzymes.
authors.affiliation: Botanisches Institut der Universität Tübingen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Rose
authors.forename: W E
authors.initials: WE
authors.lastname: Glässgen
authors.forename: W
authors.initials: W
authors.lastname: Hopp
authors.forename: H U
authors.initials: HU
authors.lastname: Seitz
mesh_terms: D000872:Anthocyanins / Q000096:biosynthesis*; D001578:Benzopyrans / Q000737:chemistry; D002478:Cells, Cultured; D005700:Galactosyltransferases / Q000302:isolation & purification; D016695:Glycosyltransferases / Q000378:metabolism*; D015394:Molecular Structure; D010430:Pentosyltransferases / Q000302:isolation & purification; D014531:Uridine Diphosphate Galactose / Q000737:chemistry; D000097608:UDP Xylose-Protein Xylosyltransferase
chemical_list: D000872:Anthocyanins; D001578:Benzopyrans; D005700:Galactosyltransferases; D016695:Glycosyltransferases; D010430:Pentosyltransferases; D014531:Uridine Diphosphate Galactose; D000097608:UDP Xylose-Protein Xylosyltransferase; C017154:cyanidin