Incorporation of UDP-[C]Glucose into Xyloglucan by Pea Membranes

Abstract

The water-insoluble 1,4-beta-linked products formed from UDP-[(14)C]glucose by pea membranes were dissolved in hot dimethyl-sulfoxide/paraformaldehyde and fractionated on columns of controlled pore glass beads calibrated with dextran standards. The products eluted with a peak size close to 70 kilodaltons in dextran equivalents. Similar elution profiles were obtained for products formed in brief or extended incubations and at high or low substrate concentrations. Methylation analysis indicated that only a few [(14)C]glucose units had been added to an endogenous acceptor to form this product. In the presence of UDP-xylose at concentrations equal to or less than UDP-[(14)C]glucose, incorporation from the latter was enhanced and the products elongated with time to a size range where the major components eluted between dextran 264 and 500 kilodaltons. Treatment with endo-1,4-beta-glucanase resulted in a mixture of oligosaccharides, including the xyloglucan subunit Glc(4)Xyl(3), which were hydrolyzed further by mixed glycosidases to labeled glucose and isoprimeverose (xylosyl-1,6-alpha-d-glucose). In pulse-chase experiments, the low molecular weight product formed from UDP-[(14)C]glucose alone was clearly a precursor for high molecular weight products formed subsequently in the presence of both UDP-glucose and UDP-xylose. It is concluded that the 1,4-beta-transglucosylation activity detected in these tests was due to an enzyme that is required for biosynthesis of the backbone of xyloglucan.