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

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 trans ported into the lumen of the Golgi by membrane-localized transporters. To s tudy the transport and metabolism of UDP-GalA in the Golgi, UDP-GalA labele d in the uridine moiety is required. Here we present a high-yield method fo r the synthesis of [H-3]UDP-GalA from [H-3]UTP and Glc-1-P by sequential re actions catalyzed by UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and UDP-GlcA-4-epimerase and the separation of the reaction products over a Dio nex CarboPac PA1 anion-exchange column using high-performance anion-exchang e chromatography (HPAEC). Approximately half of the [H-3]UTP was converted into [H-3]UDP-GalA and the remaining 50% was recovered as [H-3]UDP-GlcA. Bo th products were purified and the identity of the [H-3]UDP-GalA was confirm ed by its conversion into [H-3]UDP-GlcA by UDP-GlcA-4-epimerase. The enzyma tic synthesis of diverse nucleotide sugars radiolabeled in the nucleotide b y the use of nucleotide-converting enzymes, combined with the high-resoluti on separation of the nucleotide sugars and their purification by HPAEC, can provide unique substrates required for the study of diverse nucleotide sug ar transporters. (C) 1999 Academic Press.