Xylan synthesis from uridine-diphosphate-d-xylose by particulate preparations from immature corncobs.

Syntheses of several plant polysaccharides involving in vitro glycosyl transfer from nucleotide sugars have now been reported using the enzymes present in particulate preparations from plant tissues. Such syntheses include starch from UDP-D-glucose1 or ADP-D-glucose,2 pectin from UDP-D-galacturonic acid,3 callose from UDP-D-glucose,4 and cellulose from GDP-D-glucose5 or UDP-D-glucose.6 Indirect evidence strongly suggests that UDP-D-xylose and UDP-L-arabinose are glycosyl sources in the synthesis of the xylan fractions of plant hemicellulose. Both UDP-D-xylose and UDP-L-arabinose have been found in plants,7 and enzymes catalyzing the conversion of UDP-D-glucuronic acid to UDP-D-xylose8 9 and its subsequent epimerization to UDP-L-arabinoseO have been obtained in plant extracts. The actual transfer of xylose and arabinose from these sugar nucleotides to growing xylan polymer has not, however, been demonstrated.10 Although Pridham and Hassid" showed that corn shoot particulate preparations can transfer both D-xylose and L-arabinose from their UDP-compounds to a polymer, the possible polysaccharide formed was not characterized in detail. Since germinating mung bean shoots are not particularly rich in xylan'2 but mature corncobs (Zea mays) are known to have an unusually high content of xylan,'3 the immature, developing corncob was investigated as a source of xylansynthesizing enzymes. The presenlt paper reports the in vitro synthesis of xylan from UDP-D-xylose by particulate preparations from immature corncobs. Materials and Methods.-Radioactive UDP-D-xylose-C'4 of specific activity 106 ,uc/,umole was kindly prepared by Dr. D. S. Feingold from IJDP-D-glucuronic acid-C'4 by the action of a wheat germ decarboxylase which was free from UDP-D-xylose epimerase.9 A hydrolysate of the UDPD-xylose-Cl4 showed on paper chromatograms D-xylose-Cl4 but no detectable Larabinose-C'4. The UDP-D-glucuronic acid was prepared from a-D-glucose-C'4-1-P04 and UTP by the sequential action of mung bean pyrophosphorylase'4 and liver uridine diphosphoglucose dehydrogenase (Sigma). A mixture of UDP-D-xylose-C'4 and UDP-Larabinose-C'4 (specific activity, 106 ,uc! ,umole), designated as UDP-pentose-C'4, was prepared by the action of crude wheat germ decarboxylase'9 on UDP-D-glucuronic acid-C'4. Water-insoluble xylan (Pfanstiel, Waukegan, Illinois) which contained 5% of arabinosyl residues was purified by dissolving in 2% (w/v) alkali and reprecipitating with acetic acid and ethanol (2 vol). Water-soluble arabinoxylan, containing 13% of arabinosyl residues, was prepared from wheat flour.'6 Authentic l-1,4-xylodextrins, degree of polymerization (D.P.) = 2, 3, 4, and 5, respectively, were prepared by the partial hydrolysis of xylan with fuming HCO and subsequent fractionation of the hydrolysate on a charcoal-celite column. An aqueous solution of hemicellulase (Nutritional Biochemicals Corp.) was dialyzed at 20 before use. All other chemicals were obtained from commercial sources. Corncob particulate preparations: Sweet corn plants, variety Captain Golden, were grown in a greenhouse and immature cobs collected immediately before use. They were harvested when the actual cob within the sheath was 2-3 in. long, which was at the time the silk had just emerged from the ear. After removal of the sheath and silk, 20-50 gm of the small cobs were broken into pieces, chilled to 20, and ground in a mortar with sand and an equal weight of 0.1 M phosphate or Tris-HCl buffer (pH 7.5) containing 1% crystalline bovine serum albumin. The slurry was squeezed through cheesecloth, centrifuged at 1000 X g for 15 min to remove coarse debris and then at 30,000 X g for 30 min to sediment enzymically active particles. All operations were done