Enzymatic synthesis and purification of [(3)H]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 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 [(3)H]UDP-GalA from [(3)H]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 [(3)H]UTP was converted into [(3)H]UDP-GalA and the remaining 50% was recovered as [(3)H]UDP-GlcA. Both products were purified and the identity of the [(3)H]UDP-GalA was confirmed by its conversion into [(3)H]UDP-GlcA by UDP-GlcA-4-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.
Department of Biology, Faculty of Sciences, University of Chile, Casilla 653, Santiago, Chile.
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Orellana
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Mohnen
D002352:Carrier Proteins / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000379:methods; D006056:Golgi Apparatus / Q000378:metabolism*; D007202:Indicators and Reagents; D043564:Inorganic Pyrophosphatase; D007553:Isotope Labeling / Q000379:methods; D011755:Pyrophosphatases; D014316:Tritium; D014534:UDPglucose 4-Epimerase; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014533:Uridine Diphosphate Glucose Dehydrogenase; D014539:Uridine Diphosphate Sugars / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism*
D002352:Carrier Proteins; D007202:Indicators and Reagents; D014539:Uridine Diphosphate Sugars; D014316:Tritium; C050306:UDP-galacturonic acid; D014533:Uridine Diphosphate Glucose Dehydrogenase; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D014534:UDPglucose 4-Epimerase