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 20-25 degrees C 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)>150000) 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) 60000) beta-(1-->4)-galactan was a more efficient acceptor [specific activity 2000-3000 pmol min(-1) (mg protein)(-1)] than low- M(r) (average M(r) 10000 and 5000) 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.
Konishi
Tomoyuki
T
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Saitama 338-8570, Japan.
Mitome
Terukazu
T
Hatsushika
Hiroyuki
H
Haque
Md Ashraful
MA
Kotake
Toshihisa
T
Tsumuraya
Yoichi
Y
D002250:Carbon Radioisotopes / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism; D005685:Galactans / Q000096:biosynthesis*; D005700:Galactosyltransferases / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D018546:Hypocotyl / Q000201:enzymology; D007700:Kinetics; D031297:Lupinus; D008565:Membrane Proteins / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D009844:Oligosaccharides; D010368:Pectins / Q000096:biosynthesis*; D011506:Proteins / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*
C411147:1,4-beta-galactan; D002250:Carbon Radioisotopes; D004187:Disaccharides; D005685:Galactans; D008565:Membrane Proteins; D009844:Oligosaccharides; D011506:Proteins; C056853:6-O-galactopyranosylgalactose; D010368:Pectins; D005700:Galactosyltransferases; D006026:Glycoside Hydrolases; C094192:arabinogalactan endo-1,4-beta-galactosidase