SYNTHESIS OF (1-]3), (1-]4)-BETA-D-GLUCAN IN THE GOLGI-APPARATUS OF MAIZE COLEOPTILES

Membranes of the Golgi apparatus from maize (Zea mays L.) were used to synthesize in vitro the (1 --> 3), (1 --> 4)-beta-D-glucan (MG) that is unique to the cell wall of the Poaceae. The MG was about 250 kDa an d was separated from a much larger (1 --> 3)-beta-D-glucan (callose) b y gel-permeation chromatography. Diagnostic oligosaccharides, released by a sequence-dependent endoglucanase from Bacillus subtilis, were se parated by HPLC and GLC. The trisaccharide beta-D-Glcp-(1 --> 4)-beta- D-Glcp-(1 --> 3)-D-Glc, the tetrasaccharide [beta-D-Glcp-(1 --> 4)]2-b eta-D-Glcp-(1 --> 3)-D-Glc, and longer cellodextrin-(1 --> 3)-D-Glc ol igosaccharides were synthesized in proportions similar to those found in purified MG. Activated charcoal added during homogenization enhance d synthesis of MG, presumably by removing inhibitory compounds. The Go lgi apparatus was determined as the site of synthesis by a combination of downward and flotation centrifugations on sucrose step gradients. The rate of synthesis did not reach saturation at up to 10 mM UDP-Glc. Chelators completely abolished synthesis, but synthase activity was r estored by addition of either MgCl2 or, to a lesser extent, MnCl2. Syn thesis continued for well over 1 h; addition of KOH to raise the pH fr om 7.2 to 8.0 during the reaction increased the rate of synthesis, whi ch indicates that a transmembrane pH gradient may facilitate synthesis of MG.