Enzymatic synthesis and purification of uridine diphosphate [14C]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-14C]galacturonic acid (UDP-[14C]GalA), the nucleotide sugar substrate for homogalacturonan biosynthesis. UDP-[14C]GalA was enzymatically synthesized by 4-epimerization of commercially available UDP-[U-14C]glucuronic acid (UDP-[14C]GlcA) using a particulate preparation from radish roots. The resulting mixture of UDP-[14C]GalA and UDP-[14C]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-[14C]GalA obtained using this procedure was 16% of the starting UDP-[14C]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.
Liljebjelke
K
K
Complex Carbohydrate Research Center, University of Georgia, Athens 30602, USA.
Adolphson
R
R
Baker
K
K
Doong
R L
RL
Mohnen
D
D
D000838:Anions; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D010368:Pectins / Q000096:biosynthesis*; D010936:Plant Extracts; D019998:Racemases and Epimerases / Q000737:chemistry / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry; D014539:Uridine Diphosphate Sugars / Q000138:chemical synthesis* / Q000302:isolation & purification* / Q000378:metabolism; D014540:Uridine Diphosphate Xylose / Q000737:chemistry; D014675:Vegetables / Q000737:chemistry
D000838:Anions; D002250:Carbon Radioisotopes; D010936:Plant Extracts; D014539:Uridine Diphosphate Sugars; D014535:Uridine Diphosphate Glucuronic Acid; D014540:Uridine Diphosphate Xylose; C050306:UDP-galacturonic acid; D014537:Uridine Diphosphate N-Acetylglucosamine; D010368:Pectins; D019998:Racemases and Epimerases