Biosynthesis of pectic galactan by membrane-bound galactosyltransferase from soybean (Glycine max Merr.) seedlings

We investigated the properties of a galactosyltransferase (GalT) that is involved in the synthesis of beta-(1-->4)-galactan side chains of pectins. A membrane preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls transferred [(14)C]Gal from UDP-[(14)C]Gal into intact and partially hydrolyzed lupin beta-(1-->4)-galactans of various chain lengths as exogenous acceptors, while activity to endogenous acceptors was negligible. Maximal activity occurred at pH 6.5 and 20-25degreesC in the presence of 25 mM Mn(2+) and 0.75% Triton X-100. The transfer reaction onto the unmodified commercial pectic galactan (M(r)>150,000) from lupin we used was very low but increased when the M(r) of the galactan was reduced by partial acid hydrolysis. Among the partially hydrolyzed galactans, high-M(r) (average M(r) 60,000) beta-(1-->4)-galactan was a more efficient acceptor [specific activity 2,000-3,000 pmol min(-1) (mg protein)(-1)] than low-M(r) (average M(r) 10,000 and 5,000) polymers. Digestion of the radiolabeled product from high-M(r) galactan with endo-beta-(1-->4)-galactanase released mainly radioactive beta-(1-->4)-galactobiose and Gal, indicating that the transfer of [(14)C]Gal occurred through beta-(1-->4)-linkages. HPLC analysis showed that the enzyme also catalyzes incorporation of Gal into pyridylaminated (PA) beta-(1-->4)-galactooligomers with degree of polymerization at least 5. Gal(7)-PA chains were elongated by attachment of one, two, or three Gal residues leading to the formation of Gal(8-10)-PA.