Enzymatic Synthesis and Purification of Uridine Diphosphate [14C] Galacturonic Acid: A Substrate for Pectin Biosynthesis

✍ Scribed by K. Liljebjelke; R. Adolphson; K. Baker; R.L. Doong; D. Mohnen

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 835 KB
• Volume: 225
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1158

✦ Synopsis

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- ({ }^{14}) C]galacturonic acid (UDP- (\left[{ }^{14} \mathrm{C}\right]) GalA), the nucleotide sugar substrate for homogalacturonan biosynthesis. UDP(\left[{ }^{14} \mathrm{C}\right] \mathrm{GalA}) was enzymatically synthesized by 4 -epimerization of commercially available UDP-[U- (\left.{ }^{14} \mathrm{C}\right]) glucuronic acid (UDP- (\left[{ }^{14} \mathrm{C}\right] \mathrm{GlcA}) ) using a particulate preparation from radish roots. The resulting mixture of UDP- (\left[{ }^{14} \mathrm{C}\right]) GalA and UDP- (\left[{ }^{14} \mathrm{C}\right]) 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 \mathrm{~nm}) or by pulsed amperometric detection following postcolumn addition of (\mathrm{NaOH}). The yield of UDP- (\left[{ }^{14} \mathrm{C}\right]-) GalA obtained using this procedure was (16 %) of the starting UDP- (\left[{ }^{14} \mathrm{C}\right]) GlcA. Establishment of a reliable method to synthesize and purify UDP- (\left[{ }^{14} \mathrm{C}\right] \mathrm{GalA}) will facilitate the identification and purification of the galacturonosyltransferase(s) involved in pectin biosynthesis. & 1995 Academic Press, Inc.