ENZYMATIC-SYNTHESIS AND PURIFICATION OF URIDINE-DIPHOSPHATE [C-14] GALACTURONIC ACID - A SUBSTRATE FOR PECTIN BIOSYNTHESIS

Pectins are complex polysaccharides that contain 1,4-linked alpha-D-galactosyluronic acid residues found in the primary wall of all higher plant cells, The pectic polysaccharides play critical roles in cell wall structure and in plant growth and development. As a first step in studying pectin biosynthesis a method was developed to routinely generate and purify UDP-[U-C-14]galacturonic acid (UDP-[C-14]GalA), the nucleotide sugar substrate for homogalacturonan biosynthesis, UDP-[C-14]GalA was enzymatically synthesized by 4-epimerization of commercially available UDP-[U-C-14]glucuronic acid (UDP-[C-14]GlcA) using a particulate preparation from radish roots, The resulting mixture of UDP-[C-14]GalA and UDP-[C-14]GlcA was separated by high-performance anion-exchange chromatography using a Dionex CarboPac PA1 anion-exchange column, The UDP-sugars were detected by their absorbance at 262 nm or by pulsed amperometric detection following postcolumn addition of NaOH. The yield of UDP-[C-14]GalA obtained using this procedure was 16% of the starting UDP-[C-14]GlcA, Establishment of a reliable method to synthesize and purify UDP-[14C]GalA will facilitate the identification and purification of the galacturonosyltransferase(s) involved in pectin biosynthesis. (C) 1995 Academic Press, Inc.