Glucomannan synthesis in pea epicotyls - the mannose and glucose transferases

Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a beta-1,4-[su14C]mannan from GDP-d-[U-14C]-mannose, a mixed beta-1,3- and beta-1,4-[14C]glucan from GDP-d-[U-14C]-glucose and a beta-1,4-[14C]-glucomannan from both GDP-d-[U-14C]mannose and GDP-d-[U-14C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The beta-glucan synthase had different properties from other preparations which bring about the synthesis of beta-1,3-glucans (callose) and mixed beta-1,3- and beta-1,4-glucans and which use UDP-d-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-d-xylose in addition to GDP-d-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-d-glucose acted competitively in the presence of GDP-d-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-d-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-d-glucose and GDP-d-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-d-mannose and GDP-d-glucose to bring about the synthesis of the heteropolysaccharide.