In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish (Raphanus sativus L.) seedlings

摘要

We investigated a galactosyltransferase (GalT) involved in the synthesis of the carbohydrate portion of arabinogalactan-proteins (AGPs), which consist of a β-(1→3)-galactan backbone from which consecutive (1→6)-linked β-Galp residues branch off. A membrane preparation from 6-day-old primary roots of radish (Raphanus sativus L.) transferred [14C]Gal from UDP-[14C]Gal onto a β-(1→3)-galactan exogenous acceptor. The reaction occurred maximally at pH 5.9–6.3 and 30 °C in the presence of 15 mM Mn2+ and 0.75% Triton X-100. The apparent K m and V max values for UDP-Gal were 0.41 mM and 1,000 pmol min−1 (mg protein)−1, respectively. The reaction with β-(1→3)-galactan showed a bi-phasic kinetic character with K m values of 0.43 and 2.8 mg ml−1. β-(1→3)-Galactooligomers were good acceptors and enzyme activity increased with increasing polymerization of Gal residues. In contrast, the enzyme was less efficient on β-(1→6)-oligomers. The transfer reaction for an AGP from radish mature roots was negligible but could be increased by prior enzymatic or chemical removal of α-l-arabinofuranose (α-l-Araf) residues or both α-l-Araf residues and (1→6)-linked β-Gal side chains. Digestion of radiolabeled products formed from β-(1→3)-galactan and the modified AGP with exo-β-(1→3)-galactanase released mainly radioactive β-(1→6)-galactobiose, indicating that the transfer of [14C]Gal occurred preferentially onto consecutive (1→3)-linked β-Gal chains through β-(1→6)-linkages, resulting in the formation of single branching points. The enzyme produced mainly a branched tetrasaccharide, Galβ(1→3)[Galβ(1→6)] Galβ(1→3)Gal, from β-(1→3)-galactotriose by incubation with UDP-Gal, confirming the preferential formation of the branching linkage. Localization of the GalT in the Golgi apparatus was revealed on a sucrose density gradient. The membrane preparation also incorporated [14C]Gal into β-(1→4)-galactan, indicating that the membranes contained different types of GalT isoform catalyzing the synthesis of different types of galactosidic linkage.