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.
Orellana
A
A
Department of Biology, Faculty of Sciences, University of Chile, Casilla 653, Santiago, Chile.
Mohnen
D
D
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