Enzymatic synthesis and purification of [3H]uridine diphosphate galacturonic acid for use in studying Golgi-localized transporters

Ariel Orellana, Debra Mohnen

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

Uridine 5'-diphosphate galacturonic acid (UDP-GalA) is a substrate for the galacturonosyltransferases that synthesize the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II. Pectin synthesis occurs in the Golgi and it is hypothesized that UDP-GalA is transported into the lumen of the Golgi by membrane-localized transporters. To study the transport and metabolism of UDP-GalA in the Golgi, UDP-GalA labeled in the uridine moiety is required. Here we present a high-yield method for the synthesis of [3H]UDP-GalA from [3H]UTP and Glc-1-P by sequential reactions catalyzed by UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and UDP-GlcA-4-epimerase and the separation of the reaction products over a Dionex CarboPac PA1 anion-exchange column using high-performance anion-exchange chromatography (HPAEC). Approximately half of the [3H]UTP was converted into [3H]UDP-GalA and the remaining 50% was recovered as [3H]UDP-GlcA. Both products were purified and the identity of the [3H]UDP-GalA was confirmed by its conversion into [3H]UDP-GlcA by UDPGlcA4-epimerase. The enzymatic synthesis of diverse nucleotide sugars radiolabeled in the nucleotide by the use of nucleotide- converting enzymes, combined with the high-resolution separation of the nucleotide sugars and their purification by HPAEC, can provide unique substrates required for the study of diverse nucleotide sugar transporters.

Original languageEnglish
Pages (from-to)224-231
Number of pages8
JournalAnalytical Biochemistry
Volume272
Issue number2
DOIs
Publication statusPublished - 1 Aug 1999

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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